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Investigation of cysteine and methionine oxidation using x-ray absorption spectroscopy Karunakaran, Anusha 2010

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Investigation of Cysteine and Methionine Oxidation Using X-Ray Absorption Spectroscopy by Anusha Karunakaran B.Sc., Simon Fraser University, 2000 M.Sc., University of Sussex, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2010  © Anusha Karunakaran, 2010  ABSTRACT Cysteine (Cys) and methionine (Met) are sulfur containing amino acids with various oxidation forms. Oxidation of Cys yields cysteinyl radicals that have been postulated as intermediates in several biological contexts including enzymatic catalysis, long-range electron transfer, peptide post-translational modification and cellular redox signaling.  The challenges of detecting sulfur-based radicals with  electron paramagnetic resonance (EPR) have led to the development of Sulfur Kedge X-ray absorption spectroscopy (S K-edge XAS) as a spectroscopic tool. The reactivity of sulfur-based radicals was studied in a Pseudomonas aeruginosa azurin protein system to probe the electronic structure of isolated cysteinyl radicals, which are characterized by S 3p ← 1s pre-edge transition. S K-edge XAS has shown to be a sensitive method in detecting these cysteinyl radicals in hydrophobic and hydrophilic protein environments. The pre-edge feature of the cysteinyl radicals in hydrophobic environments was lower in energy than their hydrophilic counterparts due to hydrogen bonding interactions. Additionally, S K-edge XAS was employed to study the redox photochemistry of Met and its oxidized forms methionine sulfoxide (MetSO) and methionine sulfone (MetSO2). Met is easily photooxidized to MetSO and MetSO2 in the presence of O2. In the absence of O2, photoirradiation leads to the one-electron-oxidized Met cation radical (MetS•+), suggesting an alternative mechanism for photooxidation of thioethers through direct oxidation. The photoirradiation of MetSO leads back to Met under both aerobic and anaerobic conditions while MetSO2 is photochemically inert. These findings provide new insights into the formation of age-related cataracts. ii  Finally, the metal-induced Met oxidation in amyloid-β (Aβ) peptide was investigated. Much of the research to date has focused on the redox chemistry of Cu2+ in Aβ peptide with inconsistent findings with regards to the role of Met35 and the oxidation state of the Met35. Findings reported here indicate that in the presence of Cu2+ alone, Met35 was oxidized to MetSO, but surprisingly Fe3+ failed to oxidize the Met. These differences in the oxidation behaviour lead to the investigation of the metal binding site in Aβ. Fe3+ found to be in a six-coordinate environment with oxygen-rich ligands while Cu2+ is in a five-coordinate environment with histidine-rich ligands.  iii  TABLE OF CONTENTS Abstract .................................................................................................................... ii Table of Contents.................................................................................................... iv List of Tables ......................................................................................................... viii List of Figures ......................................................................................................... ix List of Schemes .................................................................................................... xiii List of Equations ................................................................................................... xiv Abbreviations and Symbols .................................................................................. xv Acknowledgements ............................................................................................ xviii Chapter 1: Introduction ........................................................................................ 1 1.1 Thesis Overview .......................................................................................... 1 1.2 The Chemistry of Cysteine and Methionine ................................................. 2 1.3 Reactive Oxygen and Nitrogen Species ...................................................... 4 1.4 Cysteine Oxidation ...................................................................................... 7 1.4.1  Intra- and Intermolecular Disulfides ................................................................... 8  1.4.2 1.4.3  Sulfenic Acids ..................................................................................................14 Sulfinic and Sulfonic Acids ...............................................................................19  1.4.4  S-Nitrosylation..................................................................................................22  1.4.5  Cysteinyl Radical .............................................................................................23  1.5  Methionine Oxidation ................................................................................. 28  1.5.1  Formation and Role of Sulfoxide ......................................................................28  1.5.2  Formation and Role of Sulfone.........................................................................32  1.5.3  Methionine Radical Cation ...............................................................................33  1.6  Implications of Cys and Met Oxidation in Diseases ................................... 35  Chapter 2: X-Ray Absorption Spectroscopy..................................................... 39 2.1 Background and Theory ............................................................................ 39 2.1.1  X-Ray Absorption Near-Edge Structure (XANES) ............................................41  2.1.2  Extended X-Ray Absorption Fine Structure (EXAFS) .......................................44  2.2 2.3  Experimental Set-up .................................................................................. 48 Data Reduction and Analysis ..................................................................... 52  2.3.1  Sulfur K-edge XANES ......................................................................................52  2.3.2  Metal K-edge XANES and EXAFS ...................................................................53  iv  Chapter 3: Synthesis and Characterization of Cysteinyl Radicals in Pseudomonas Aeruginosa Azurin........................................................................ 56 3.1 Introduction ................................................................................................ 56 3.1.1  Implications of Cysteinyl Radicals in Biology ....................................................56  3.1.2  Current Spectroscopic Detection ......................................................................57  3.1.3  Proposed Spectroscopic Detection ..................................................................59  3.1.4 3.1.5  Pseudomonas aeruginosa Azurin.....................................................................61 Cysteines of Interest in Two Distinct Regions of Azurin....................................62  3.2  Experimental.............................................................................................. 64  3.2.1  Multi Site-directed Mutagenesis .......................................................................64  3.2.2  Sequencing ......................................................................................................65  3.2.3  Protein Expression and Purification..................................................................65  3.2.4 3.2.5  Synthesis of Rhenium Complex .......................................................................69 Re-labeling Reaction ........................................................................................71  3.2.6  Purification of Labeled Azurins .........................................................................71  3.2.7  The Flash/Quench/Freeze Methodology ..........................................................72  3.2.8 3.2.9  XAS Data Acquisition .......................................................................................73 XAS Data Processing and Analysis..................................................................73  3.3  Results and Discussion ............................................................................. 75  3.3.1  Re-labeled Azurin mutants ...............................................................................75  3.3.2  Generation of Cysteinyl Radicals .....................................................................77  3.3.3  Characterization of Cysteinyl Radicals by Sulfur K-edge XAS ..........................78  3.4  Conclusions ............................................................................................... 82  Chapter 4: Photochemistry of Methionine, Methionine Sulfoxide, and Methionine Sulfone ................................................................................................ 83 4.1 Introduction ................................................................................................ 83 4.1.1  Methionine Oxidation .......................................................................................83  4.1.2  Age-related Cataracts ......................................................................................84  4.1.3  Photooxidation of Thioether .............................................................................85  4.2  Experimental.............................................................................................. 87  4.2.1 4.2.2  Materials ..........................................................................................................87 Sample Preparation .........................................................................................87  4.2.3  XAS Data Acquisition .......................................................................................88  4.2.4  Data Processing and Analysis ..........................................................................88  4.3  Results and Discussion ............................................................................. 89  4.3.1  Met Photochemistry .........................................................................................89  4.3.2  MetSO Photochemistry ....................................................................................95  v  4.3.3  4.4  MetSO2 Photochemistry .................................................................................101  Conclusions ............................................................................................. 102  Chapter 5: Metal-Induced Methionine Oxidation in Amyloid-Beta Peptide .. 104 5.1 Introduction .............................................................................................. 104 5.1.1  Alzheimer’s Disease.......................................................................................104  5.1.2  Amyloid-beta (Aβ) Peptide .............................................................................105  5.1.3  Metal Ion Involvement in Alzheimer’s Disease ...............................................106 The Role of Met35 in Aβ ..................................................................................110  5.1.4  5.2  Experimental............................................................................................ 112  5.2.1  Materials ........................................................................................................112  5.2.2  Sample Preparation .......................................................................................113  5.2.3  XAS Data Acquisition .....................................................................................114  5.2.4  Data Processing and Analysis ........................................................................114  5.3 5.3.1 5.3.2  5.4  Results and Discussion ........................................................................... 114 Copper(II)-Induced Oxidation .........................................................................114 Iron(III)-Induced Oxidation .............................................................................119  Conclusions ............................................................................................. 121  Chapter 6: Structural Characterization of the Copper(II) and Iron(III) Coordination to the Amyloid-Beta Peptide ........................................................ 122 6.1 Introduction .............................................................................................. 122 6.1.1 6.1.2  Experimental Techniques ...............................................................................122 Aβ-Cu Complexes ..........................................................................................123  6.1.3  Aβ-Zn Complexes ..........................................................................................124  6.1.4  Aβ-Fe Complexes ..........................................................................................124  6.2  Experimental............................................................................................ 125  6.2.1  Materials ........................................................................................................125  6.2.2 6.2.3  Sample Preparation .......................................................................................125 XAS Data Acquisition .....................................................................................126  6.2.4  Data Processing and Analysis ........................................................................126  6.2.5  Model Building and Fitting Strategy ................................................................127  6.3  Results and Discussion ........................................................................... 129  6.3.1  AβCu2+ Complexes ........................................................................................129  6.3.2  AβFe3+ Complexes .........................................................................................137  6.4  Conclusions ............................................................................................. 143  vi  Chapter 7:  Concluding Remarks and Future Directions ................................ 145  References ........................................................................................................... 150 Appendix A: DNA Sequencing Output .............................................................. 164 Appendix B: Rhenium-Labeled Azurin Data ..................................................... 165 Appendix C: List of Publications ....................................................................... 167  vii  LIST OF TABLES Table 2.1  Summary of structural information extracted from EXAFS. ..............................47  Table 2.2  Calculated and refined parameters in the EXAFS fitting process. ....................55  Table 3.1  Additional mutations in Y108Cm and W48Cm. ...................................................64  Table 3.2  Protein expression conditions attempted for Y108Cm and W48Cm. The highlighted condition produced observable protein expression level. ...............76  Table 3.3  Mass spectral results for the labeled and unlabeled azurin mutants. ................77  Table 4.1  Results from principle component analysis of in situ photoirradiation of MetSO by Xe arc lamp under anaerobic conditions. The highlighted portion shows the minimum number of components needed for >99% cumulative variance. ........99  Table 4.2  Results of PCA target transformation performed in SixPACK ...........................99  Table 6.1  EXAFS parameters for Aβ1-16Cu2+ and Aβ1-40Cu2+. .........................................132  Table 6.2  EXAFS parameters for Aβ1-16Fe3+ and Aβ1-40Fe3+. ..........................................141  viii  LIST OF FIGURES Figure 1.1  Structures of cysteine and methionine........................................................... 3  Figure 1.2  Bertrand diagram indicating the benefit of ROS/RNS depends on its concentrations. .............................................................................................. 6  Figure 1.3  Common oxidative modifications of cysteine in proteins that have been observed or proposed to occur in vivo. (Formal oxidation states for the sulfur atom(s) in these species are given in parentheses.) ...................................... 8  Figure 1.4  Mechanism of PTP activity regulated by H2O2. Y is tyrosine residue in protein and receptor. YP and PY are phosphorylated tyrosine residues.107 .............19  Figure 1.5  Formation of cysteine oxidation products in proteins and peptides. Irreversible oxidation products are framed with dashed lines. The dashed arrow represents one exception where occurs in Prx. ..................................27  Figure 1.6  Common oxidative modifications of methionine in proteins that have been observed or proposed to occur in vivo. (Formal oxidation states for the sulfur atom in these species are given in parentheses.) .........................................28  Figure 2.1  XAS spectrum depicting XANES and EXAFS regions. .................................40  Figure 2.2  XANES transitions and spectral features. .....................................................42  Figure 2.3  Sulfur K-edge spectra of series of oxidized sulfur species. ...........................43  Figure 2.4  The constructive and destructive interference of photoelectron waves that give rise to EXAFS. ......................................................................................44  Figure 2.5  Scattering pathways in a three-body configuration consisting of absorber (A) and scatterer (S). (I) is the single-scattering pathway and (II) and (III) are possible multiple-scattering pathways. .........................................................48  Figure 2.6  Schematic of a synchrotron ..........................................................................49  Figure 2.7  XAS experimental set-up..............................................................................50  Figure 2.8  EXAFS data processing: A) spline function, B) k3-space and C) R-space plots. ............................................................................................................54  Figure 3.1  First derivative X-band EPR spectra of cysteinyl radicals. A) RNR protein of E. coli (500μM); B) BSA (4 mM); and C) cysteine (300 mM). This figure has been reproduced with permission.236 ............................................................59  Figure 3.2  Qualitative valence molecular orbital diagram depicting electron excitation of sulfur species: sulfide anion, thiolate and thiyl radical..................................60  ix  Figure 3.3  Azurin model system highlighting the different electronic environments. A is hydrophilic, B is hydrophobic and C is metal binding region. ........................62  Figure 3.4  Re-photoinitiator complex.............................................................................70  Figure 3.5  SDS-PAGE Gel. Lane 1 is protein ladder, lanes 2-7 are purified fractions of Y108C, and lane 8 is Y108C isolated by osmotic shock. ..............................75  Figure 3.6  Simplified flash/quench reaction between Re-complex and the nearby Cys with oxidative quencher (Qo). .......................................................................78  Figure 3.7  S K-edge XAS spectra of W48C and W48C radical with pre-edge feature expanded (inset). .........................................................................................80  Figure 3.8  S K-edge XAS spectra of Y108C and Y108C radical with pre-edge feature expanded (inset). .........................................................................................81  Figure 3.9  S K-edge XAS experimental data and fit for W48C radical. ..........................81  Figure 4.1  Sample compartment for in situ anaerobic photochemistry setup at SSRL beamline 6-2, where I0 is the incident X-ray beam. .......................................88  Figure 4.2  S K-edge XAS spectra with photoirradiation of Met by Xe arc lamp under aerobic conditions. .......................................................................................90  Figure 4.3  S K-edge XAS spectra of Met, MetSO, MetSO2, CH3SO3-, and SO4-2. ..........91  Figure 4.4  S K-edge XAS spectra with in situ photoirradiation of Met by Xe arc lamp under anaerobic condition. Inset shows the difference spectra of the lowenergy. .........................................................................................................92  Figure 4.5  Kinetic profile of the weak pre-edge feature from in situ photoirradiation of Met by Xe arc lamp under anaerobic conditions. ..........................................93  Figure 4.6  UV-Vis spectra of Met, MetSO, and MetSO2. ...............................................94  Figure 4.7  S K-edge XAS spectra with photoirradiation of MetSO by Xe arc lamp under aerobic conditions. .......................................................................................95  Figure 4.8  S K-edge XAS spectra with in situ photoirradiation of MetSO with Xe arc lamp under anaerobic conditions. .........................................................................96  Figure 4.9  S K-edge XAS spectra with X-ray photoreduction of MetSO during data  Figure 4.10  1  collection. .....................................................................................................97 H NMR spectra of MetSO photoirradiation with Xe arc lamp products under  anaerobic conditions after 15 minutes. .........................................................98 Figure 4.11  Linear least-squares fit of in situ MetSO photoirradiation by Xe arc lamp under anaerobic conditions after 90 minutes. .............................................100  x  Figure 4.12  S K-edge XAS spectra with in situ photoirradiation of DMSO by Xe arc lamp under anaerobic conditions. .......................................................................101  Figure 4.13  S K-edge XAS spectra with photoirradiation of MetSO2 by Xe arc lamp under aerobic conditions. .....................................................................................102  Figure 5.1  Processing of the amyloid precursor protein (APP) to yield amyloid-β (Aβ) peptide. ......................................................................................................105  Figure 5.2  Amino acid sequence of Aβ1-42. Note that the peptide contains a single methionine residue at position 35. ..............................................................106  Figure 5.3  Schematic view of the amyloid cascade hypothesis.301 ..............................108  Figure 5.4  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Cu2+/H2O2 or H2O2. .....................................................................................115  Figure 5.5  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Cu2+. ...................................................................................................................116  Figure 5.6  S K-edge XAS spectra of N-acetyl-methionine amide (ac-Met-NH2) before and after incubation with Aβ1-16Cu2+. ..........................................................118  Figure 5.7  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Fe3+/H2O2 or H2O2. .....................................................................................120  Figure 5.8  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Fe3+. ...................................................................................................................120  Figure 6.1  The XANES region of the Cu K-edge XAS of AβCu2+. The pre-edge transition of 3d ← 1s is marked a and b represents the 4p ← 1s + shakedown transition. ...................................................................................................130  Figure 6.2  EXAFS of Aβ1-16Cu2+and Aβ1-40Cu2+. A) and B) are k3 weighted χ(k) of Aβ116Cu  2+  and Aβ1-40Cu2+, respectively. C) and D) are corresponding Fourier-  transformed (FT) of Aβ1-16Cu2+ and Aβ1-40Cu2+. Experimental data are shown by dashed lines and simulations to the data by solid lines. .........................133 Figure 6.3  Schematic picture of the molecular arrangement around the Cu2+ in Aβ. Atom colours are as follows: copper (Cu) black; oxygen (O) red; nitrogen (N) blue; carbon (C) gray. ................................................................................134  Figure 6.4  Four-coordinate and six-coordinate fits of Aβ1-40Cu2+. A) and B) are k3 weighted χ(k) of Aβ1-40 with four-coordinate and six-coordinate Cu2+ fits, respectively. C) and D) are corresponding fits in R-space. Experimental data are shown by dashed lines and simulations to the data by solid lines.........135  xi  Figure 6.5  EXAFS of Cu2+ in buffer. A) is k3 weighted χ(k) of Cu2+ in buffer and B) is the corresponding Fourier-transformed (FT) of Cu2+ in buffer Experimental data are shown by dashed lines and simulations to the data by solid lines.........137  Figure 6.6  The XANES region of the Fe K-edge XAS of AβFe3+. Inset shows the Fe 3d ← 1s pre-edge features..............................................................................139  Figure 6.7  EXAFS of Aβ1-16Fe3+ and Aβ1-40Fe3+. A) and B) are k3 weighted χ(k) of Aβ13+ 16Fe  and Aβ1-40Fe3+, respectively. C) and D) are corresponding Fourier  transformed (FT) of Aβ1-16Fe3+ and Aβ1-40Fe3+. Experimental data are shown by dashed lines and simulations to the data by solid lines. .........................140 Figure 6.8  Schematic picture of the molecular arrangement around the Fe3+ in Aβ. Atom colours are as follows: iron (Fe) black; oxygen (O) red; nitrogen (N) blue; carbon (C) gray. .........................................................................................141  Figure 6.9  Five-coordinate fit of Aβ1-40Fe3+. A) k3 weighted χ(k) of Aβ1-40 with fivecoordinate Fe3+ fit. B) is corresponding fit in R-space. Experimental data are shown by dashed lines and simulations to the data by solid lines. ..............142  Figure 6.10  EXAFS of Fe3+ in buffer. A) is k3 weighted χ(k) of Fe3+ in buffer and B) is the corresponding Fourier-transformed (FT) of Fe3+ in buffer Experimental data are shown by dashed lines and simulations to the data by solid lines.........143  Figure A.0.1 DNA sequence of Y108Cm ...........................................................................164 Figure A.0.2  DNA sequence of W48Cm ..........................................................................164  Figure B.0.3  MALDI-TOF result of Re-labeled Y108C and unlabeled Y108C before purification..................................................................................................165  Figure B.0.4  MALDI-TOF result of Re-labeled W48C and unlabeled W48C before purification..................................................................................................166  xii  LIST OF SCHEMES Scheme 1.1  Generation and interconversion of ROS ........................................................ 5  Scheme 1.2  Generation and interconversion of RNS ........................................................ 5  Scheme 1.3  Reduction of protein disulfide bond by thioredoxin/thioredoxin reductase system.65 PSSP is interprotein disulfide bond and PSH is the reduced protein .....................................................................................................................10  Scheme 1.4  Formation of protein S-glutathionylation, A) Thiol-disulfide exchange, B) Cysteinyl radical, C) Cysteine sulfenic acid, D) Thiosulfinate, E) Snitrosylcysteine.............................................................................................12  Scheme 1.5  Mechanism of GRx-catalyzed protein deglutathionylation.80 .........................12  Scheme 1.6  Stepwise reduction of thiosulfinate. ..............................................................14  Scheme 1.7  Formation of cysteine sulfenic acid...............................................................15  Scheme 1.8  Formation of cyclic sulfenamide. ..................................................................16  Scheme 1.9  Mechanism of 1-Cys and 2-Cys peroxiredoxins.102 .......................................18  Scheme 1.10 Proposed mechanism of sulfiredoxin catalyzed sulfinic acid in peroxiredoxin.119 ...........................................................................................21 Scheme 1.11  Reactions of cysteinyl radicals..................................................................25  Scheme 1.12  Proposed mechanism of bovine methionine sulfoxide reductase.156 .........30  Scheme 1.13  Proposed mechanism for E.coli methionine sulfoxide reductase.157 ..........31  Scheme 1.14  One electron oxidation of N-acetyl-L-methionine amide............................34  Scheme 4.1  Oxidation and reduction of methionine residues in proteins. .........................83  Scheme 4.2  Proposed mechanism of photooxidation of thioether involves the formation of singlet oxygen.259 .........................................................................................86  Scheme 4.3  Alternative mechanism of photooxidation of thioether involves formation of sulfide radical cation.261 ................................................................................86  Scheme 4.4  Met photooxidation by Xe arc lamp and photoreduction by X-ray beam. ......93  Scheme 5.1  Proposed mechanism of H2O2 production by Aβ bound to redox active metal.304 .....................................................................................................110  xiii  LIST OF EQUATIONS Equation 1.1  Thiol-disulfide exchange reaction .............................................................. 9  Equation 2.1  Photoelectron wavenumber, k ..................................................................45  Equation 2.2  EXAFS single-scattering equation ............................................................46  Equation 2.3  Bragg's Law .............................................................................................50  Equation 2.4  Oscillatory fine structure, χ (E ) ................................................................53  Equation 2.5  R-factor ....................................................................................................55  Equation 3.1  Voigt Area Function ..................................................................................74  Equation 3.2  Cumulative Gaussian/Lorentzian Function ...............................................74  Equation 5.1  Fenton chemistry. ...................................................................................109  Equation 5.2  Haber-Weiss reaction. ............................................................................109  xiv  ABBREVIATIONS AND SYMBOLS = ± ~ ← δ δa(k) ∆ εr  σi2 µ µ(E) µ0(E) ν θ χ(E) λ λ(k) φi(k) ψi(k) A Å Ai(k) Aβ AD ac-Met-NH2 APP BSA BSS CaM cm-1 CN Cys Cys-SOH d D DAO dmphen DNA E E0 EDTA  minus; covalent bond equals; double bond plus or minus approximately to (indicates transition) chemical shift in ppm (NMR) absorberphase shift functions (EXAFS) change in dielectric constant mean-square deviation in Ri(EXAFS) absorption; dipole moment, D total absorption coefficient (XAS) smooth atomic background (spline function; XAS) frequency angle in degrees chi = oscillatory fine structure (EXAFS) wavelength photoelectron mean free path function (EXAFS) total EXAFS phase shift function (EXAFS) scattererphase shift functions (EXAFS) absorber atom angstrom backscattering amplitude function (EXAFS) amyloid-β Alzheimer’s disease N-acetyl-L-methionine amide amyloid precursor protein bovine serum albumin benzylsuccinate synthase calmodulin wavenumber(s) coordination number cysteine sulfenic acid lattice spacing Debye (dipole moment units) D-amino acid oxidase dimethylphenanthroline deoxyribonucleic acid incident photon energy (XAS) ionisation (threshold) energy (XAS; see also IE) ethylenediaminetetraacetic acid xv  EGF EPR ERK ESR eV EXAFS FEFF fi(k) GRx GSH GSNO GSOH GSSG h  HPLC HSA HIV HOTf I0 I1 I2 IFEFFIT IPTG JNK k LB m Met MetS•+ Met-SO min. MS Msr MWCO n Ni NADPH NAPS NHase NMR OD OTfPBS PC PCA  epidermal growth factor electron paramagnetic resonance extracellular signal regulated kinase electron spin resonance electronvolt(s) extended x-ray absorption fine structure ab initio code (EXAFS) EXAFS amplitude function (EXAFS) glutaredoxin glutathione S-nitrosoglutathione glutathione sulfenic acid glutathione disulfide Planck’s constant h/2π high performance liquid chromatography human serum albumin human immunodeficiency virus triflic acid incident light transmitted light (after sample; XAS) transmitted light (after reference; XAS) command line program (EXAFS) isopropyl-β-thiogalacto-pyranoside c-Jun NH2-terminal kinase photoelectron wavenumber Luria-Bertani medium mass of the photoelectron methionine methionine radical cation methionine sulfoxide minute(s) multiple scattering methionine sulfoxide reductase molecular weight cut-off harmonic order number of atom i scatterers (EXAFS) nicotinamide adenine dinucleotide phosphate (reduced form) nucleic acid protein service nitrile hydratase nuclear magnetic resonance optical density triflate anion = CF3SO3– phosphate buffered saline principle component principle component analysis xvi  PD PDI PeakFit PFL pKa Prx PSH PSOH PSSP PSSG PTK PTP Qo R Ri RNR RNS ROS s S S02 Si(k) SHE SixPACK SOD SOD1 Srx SS SSRL TB Trx TrxR UBC UV Vis XANES XAS Z  Parkinson’s disease protein disulfide isomerase software for fitting data pyruvate formate lyase -log of the acid dissociation constant peroxiredoxin protein with Cys residue protein sulfenic acids disulfides between 2 proteins S-glutathionylated protein protein tyrosine kinase protein tyrosine phosphatase Oxidative quencher interatomic distance absorber-backscatterer distance for atom i (EXAFS) ribonucleotide reductases reactive nitrogen species reactive oxygen species second(s) Scatterer atom total amplitude reduction factor (EXAFS) amplitude reduction function (EXAFS) standard hydrogen electrode graphical user interface for XAS data processing and analysis superoxide dismutase copper-zinc superoxide dismutase sulfiredoxin single scattering Stanford Synchrotron Radiation Lightsource terrific broth (media) thioredoxin thioredoxin reductase University of British Columbia ultra-violet visible x-ray absorption near-edge structure x-ray radiation spectroscopy atomic number (atom identity for EXAFS)  xvii  ACKNOWLEDGEMENTS I would like to express my sincere gratitude first and foremost to Pierre for being an excellent supervisor. I really appreciate his flexibility and the freedom that he gave me to adapt projects in the direction that I wanted. Thank you for always having time to discuss research and other interesting topics. Your support, patience, encouragement, and understanding has been vital in my experience as a graduate student. Joining a new start-up lab has been a great experience because of Vlad, Mario, and Kendra. Thank you for your company and friendship which have made the grad school experience enjoyable. I am very thankful that you have created the right atmosphere in the lab.  I want to thank Vlad for keeping me entertained  especially during the trips to Stanford. It was great to bounce off ideas with him and I am thankful for his feedback in my research and in preparation of this thesis. I also want to thank Mario for giving a helpful hand whenever I needed, especially developing a program that was essential in my EXAFS analysis. Finally joining our core initial group is the organized Kendra, I am grateful that she was always willing to give a hand in the lab and sharing lab duties. There are plenty of things for which to thank you guys, but above all, I am thankful for our friendship and the bond we now have. I was fortunate to work with three best summer and co-op students Wendy, Alison, and Stephanie. You guys were a pleasure to supervise, and helped me so much with molecular biology work in this thesis. I also want to thank the  xviii  present group members Danielle, Thamy, Tulin, and Wei, although it was a short overlap, but it was a memorable one. I thank Dr. William Wehbi and Prof. Harry Gray for kindly providing me the mutant plasmid to start the Azurin work. I want to thank Dr. Jinhe Pan for synthesis of the amyloid-β peptide. I would also like to thank the UBC Chemistry department support staff in particular Elena Polishchuk, Candice Martin and Jie Chen for accommodating me in Biological Services, John Ellis for his help with acquisition orders, Pat Olsthoom for his help with sample shipments to SSRL and Marshall Lapawa for the mass spec data collection. Special thanks to Anka Lekhi for having a positive impact and giving great feedback on my teaching skills. I would like to express my appreciation to the staff at the Stanford Synchrotron Radiation Lightsource (SSRL) for technical and scientific support during data collection. Special thanks to Dr. Serena DeBeer Geroge, who always been ready to help at any time and Dr. Matthew Lattimer for his help with the experimental setup for our photo-irradiation studies. Finally, I would like to thank my family - Mom and Dad for encouraging and supporting in all my decisions and my grandmother for providing me with her special after-walk snacks and our tea-time which I took for granted during my years at living home. I thank Satesh for being the best brother and always pushing me to the next level. Of course special thanks go to my best biochemistry lab partner and my best friend, my husband Neil for his enduring support and tolerance. He was there for me  xix  throughout the lows and the highs and patiently listening to me rant and rave about everything. I am grateful for all his valuable lessons and always seeing the bigger picture. I feel lucky to have a great loving family and friends. To all of them go my sincerest thanks, respect and appreciation. This research is funded by NSERC (Canada).  Start-up funds and  infrastructure support were provided by UBC. Portions of this research were carried out at SSRL, a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environment Research, and by the National Institutes of Health, National Center for Research Resources Biomedical Technology Program.  xx  CHAPTER 1: INTRODUCTION  1.1  Thesis Overview This thesis seeks to address fundamental questions regarding the redox  chemistry and biochemistry of cysteine (Cys) and methionine (Met), two sulfurcontaining natural amino acids. The work presented herein explores the use of X-ray absorption spectroscopy (XAS) as a tool to probe the redox state of these two important biological molecules and, in doing so, provides new insights into the relevance of sulfur redox processes in disease, particularly in age-related cataracts and Alzheimer’s disease. The present chapter introduces the Cys and Met oxidation in biology highlighting the wealth of oxidative modifications of these two amino acids and their biological implications. It summarises the level of understanding that exists in the current scientific literature. Chapter 2 provides the background and theory of XAS. The data analysis procedures and experimental setup are also described in this chapter. In the subsequent chapters (chapter 3-6), applications of this spectroscopic technique to specific topics have been discussed. This thesis covers three distinct projects that focus on Cys and Met oxidation. Chapter 3 covers the first project which describes the synthesis and characterization of cysteinyl radical.  Currently, there is a lack of a sensitive  detection method for cysteinyl radicals. Therefore, sulfur K-edge XAS was used in  1  this project as a spectroscopic method to investigate cysteinyl radicals in distinct hydrophilic and hydrophobic protein environments. Chapter 4 describes the second project that focused on the photochemistry of methionine, methionine sulfoxide, and methionine sulfone and their importance in age-related cataract formation. The final project is divided into two chapters, chapter 5 and 6. Chapter 5 describes the work on the metal-induced methionine oxidation in amyloid-β peptide and chapter 6 describes the copper(II) and iron(III) binding in amyloid-β peptide. Finally, chapter 7 provides a conclusion of the work herein and considers future directions.  1.2  The Chemistry of Cysteine and Methionine Cys and Met are the two naturally occurring sulfur containing amino acids  typically found in peptides and proteins (Figure 1.1). In humans, Met is an essential amino acid, obtained by dietary intake while Cys is non-essential and a metabolite of Met metabolism. The Cys side chain is distinguished by a thiol functional group. The large, polarizable sulfur atom in a thiol is electron-rich and quite nucleophilic; its nucleophilicity is enhanced in its deprotonated thiolate form.  The thiol group is  mildly acidic and its pKa is dependent on the structure and local environment. The pKa of free Cys in aqueous solution is ~8 but varies considerably in proteins, ranging from as low as 3.5 to a high of 8.7.1,2 The Met side chain contains a terminal methyl group to form a dialkyl thioether.  For this reason, Met possesses a unique  combination of hydrophobic properties provided by the terminal methyl group and nucleophilic reactivity provided by the two lone pairs on the sulfur atom. Together Cys and Met fulfill a wide range of essential biological functions.  2  OH O  OH SH  NH2 Cysteine (Cys)  Figure 1.1  S  O  CH3  NH2 Methionine (Met)  Structures of cysteine and methionine  Cys residues are involved in a number of proteins and enzymes, playing both catalytic and structural functions. Catalytic Cys residues are seen in the active sites of proteases, phosphatases, and proteinases.3-5 More indirectly, Cys residues are often found as ligands to catalytically active transition metal centres, as observed in nitrile hydratase6, aconitase7, and some heme enzymes such as cytochrome P4508. The structural role of Cys is observed in protein disulfide bonds9 as well as in Zn fingers10.  In addition, Cys also plays a role in cell signaling and antioxidant  defence.9,11,12 Met plays a mandatory role as an initiator in protein synthesis. Met can also play an important role in stabilizing protein structure, although in a different manner to Cys; due to its hydrophobic properties, Met residues are usually located in the hydrophobic core of proteins, thus stabilizing their tertiary structure.13 In addition to their structural role, Met residues are found as ligands to metal centres, as seen in cytochrome c, azurin and plastocyanin.14-17 Similarly to Cys, Met also plays a role in antioxidant defence18 and cell signaling.19 Of these various functions, antioxidant defence and cell signaling rely on the rich reduction-oxidation (redox) chemistry of  3  Cys and Met, generally initiated by reactive oxygen and nitrogen species (ROS/RNS).  1.3  Reactive Oxygen and Nitrogen Species Reactive oxygen species form as a natural by-product of the normal  metabolism of dioxygen.  ROS include singlet oxygen (1O2), hydrogen peroxide  •  •  •  (H2O2), superoxide (O2 -), hydroperoxyl radical (HO2 ), hydroxyl radicals ( OH), hypochlorous acid (HOCl), chloroamine (RNHCl), and related species.20-22  A  general scheme depicting the generation and metabolism of ROS is given in •  Scheme 1.1. Mitochondrial respiration is a major source of O2 -, although enzymatic reactions such as those of NADPH oxidase and xanthine oxidase are also •  •  important.23-25 Although O2 - is a strong oxidant, its conjugate acid (HO2 ) is even stronger.26,27  •  H2O2 and O2 are formed by dismutation of O2 - catalyzed by  superoxide dismutases (SOD).28  H2O2 may be converted by neutrophil  myeloperoxidase or eosinophil peroxidise to a more reactive oxidant HOCl, which can further react with an amine to form chloroamine (RNHCl).29 produced by ionizing and UV radiation.  For example,  ROS are also  1  O2 is formed upon  photoexcitation of endogenous photosensitizers and energy transfer to ground state 3  O2.22 Also, ROS attack on metalloproteins frequently results in the release of their  metal ions, such as Zn2+, Fe2+/3+, and Cu1+/2+. The redox active iron and copper are •  able to participate in Fenton-type reactions to reduce H2O2 to form OH.30  4  O2  Oxidases Mitochondria  O2  hυ  SOD  Fenton Rxn  H2O2  OH  Peroxidase  1  HO2  O2  HOCl Amine (RNH2) RNHCl  Scheme 1.1  Generation and interconversion of ROS  •  Analogous to ROS, RNS include nitric oxide radicals (NO ) and NO-derived species such as peroxynitrite (ONOO-) and dinitrogen trioxide (N2O3). The main •  •  source of NO is via enzymatic oxidation of L-arginine by NO synthase.31 NO is unstable in an oxygen environment, therefore it can further react with O2 to give N2O3 and with O2- to give ONOO- (Scheme 1.2).32  N2O3 OH  O2  NH  O  N  NH2  NOS  NO  NH2 Arginine  O2 ONOO  Scheme 1.2  Generation and interconversion of RNS  5  ROS and RNS play an integral role in the modulation of several physiological functions but can also be potentially destructive if produced in excessive amounts. ROS and RNS are subject to the limitations of the Bertrand diagram33 (Figure 1.2). Moderate levels of ROS and RNS are essential for normal cell function such as defence against infectious agents, maturation of cellular structures, cell signaling pathways, and the induction of mitogenic response.34-38 When ROS and RNS levels exceed the cellular antioxidant capacity, a deleterious condition known as oxidative/nitrosative stress occurs.  It is a status in which cellular antioxidant  defences are insufficient to keep the levels of ROS/RNS below their toxic threshold. This can lead to DNA damage39, lipid peroxidation40, and protein modification via oxidation of amino acids41.  Oxidation of amino acids is associated with either  irreversible loss or reversible regulation of protein function.  Figure 1.2  Bertrand diagram indicating the benefit of ROS/RNS depends on its concentrations.  6  To cope with oxidative and nitrosative stress, cells have developed defence strategies at the level of damage repair and antioxidant defence. Key antioxidants are glutathione (GSH), Vitamins C and E, β-carotene, proteins such as albumin as well as enzymatic ROS-metabolizing systems including superoxide dismutase (SOD), catalase, peroxiredoxin (Prx) and GSH peroxidase.42-44 Repair mechanisms include various protein disulfide reductase enzymes as well as other reducing enzymes such as thioredoxin reductase (TrxR), methionine sulfoxide reductase (Msr) and sulfiredoxin (Srx).45-49 When ROS and RNS are not tightly controlled by these defence mechanisms, oxidative and nitrosative stress plays a role in the development of chronic and degenerative ailments such as cancer, autoimmune disorders,  rheumatoid  arthritis,  cataract,  aging,  cardiovascular  and  neurodegenerative diseases.34-37,50-53  1.4  Cysteine Oxidation Cys has long been recognized as being easily oxidized in vivo.  The  polarizable sulfur atom in Cys is subject to numerous oxidative post-translational modifications in the cellular milieu as shown in Figure 1.3. Oxidation of Cys within proteins may result in various changes to the protein’s structure and function, not all of which are detrimental. Oxidative activation and inactivation of proteins can be biologically reversible or irreversible, depending on the Cys oxidation state that is formed.  7  R  O  H N  R'  R  SH  R  R  R'  R  O R'  R  N H  R  R'  O  H N  N  H N  R  H N  Sulfenylamide (0)  O R'  R  N H  R  H N  R' O S  OH  Cysteine Sulfonic Acid (+4)  R' O  R  R'  R  Thiosulfinate (+1,-1)  O  O  O  O  R'  S  H N  S S  O Disulfide (-1)  Figure 1.3  R'  S O OH Cysteine Sulfinic Acid (+2)  O R'  O  S-nitrosylcysteine (0)  S S R  H N  S NO  Cysteinyl Radical (-1)  S OH Cysteine Sulfenic Acid (0) H N  O  S  Cysteine (-2) H N  H N  N H  O R' O S O S R' O  Thiosulfonate (+3,-1)  Common oxidative modifications of cysteine in proteins that have been observed or proposed to occur in vivo. (Formal oxidation states for the sulfur atom(s) in these species are given in parentheses.)  1.4.1 Intra- and Intermolecular Disulfides The most extensively studied oxidative modification of Cys involves the formation of intramolecular and intermolecular disulfide bonds. The formation of intramolecular disulfide bonds stabilizes the conformation of proteins and is a crucial element of maintaining the tertiary structure of proteins. The disulfide-linked folding in proteins is assisted by protein disulfide isomerase (PDI), a protein that can  8  catalyze disulfide formation and reduction as well as isomerisation of incorrect disulfides.54  PDI is found in the endoplasmic reticulum of eukaryotes.  In  prokaryotes, several periplasmic proteins are involved in protein disulfide formation; such proteins include DsbA, DsbB and DsbC.55-57 Both disulfide bond formation and isomerase activities occur by thiol-disulfide exchange reactions (Equation 1.1). The ability of PDI and DsbA to catalyze thiol-disulfide exchange reaction is correlated with a presence of a Cys-Xaa1-Xaa2-Cys motif in the active site.58  When the  cysteines in the active site are present in the disulfide form, PDI or DsbA can directly oxidize thiol groups of target proteins into disulfide bridges. The reduced DsbA is then reoxidized by DsbB59 whereas PDI is reoxidized by a protein called Ero1.60 In contrast, the isomerase activity of PDI relies on the reduced form of the cysteines in the active site, which is suitable for disulfide reshuffling.61 The isomerase activity in prokaryotes is assisted by DsbC.62 nucleophilic substitutions.  Thiol-disulfide exchange reactions are  A thiol or thiolate act as a nucleophilic agent on a  disulfide bond. These reactions are not only used to form and destroy structural disulfides in proteins but also to regulate enzyme activity, control cellular signaling pathways and to maintain the cellular redox balance. Equation 1.1 Thiol-disulfide exchange reaction  RSSR + R ' SH  R ' SSR + RSH  Intermolecular disulfides can form between two proteins (PSSP), which can have regulatory functions. For example, type I protein kinase A contains protein thiols that operate as redox sensor, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular H2O2.63 In order to serve as a  9  redox sensor, disulfide formation must be reversible. In addition to PDI, glutaredoxin (GRx) and thioredoxin (Trx) are involved in reduction of disulfides, however, Trx is more effective in reducing the disulfides in proteins.64 Trx also contains the CysXaa1-Xaa2-Cys motif in the active site, which is involved in reducing disulfide bonds in target proteins through thiol-disulfide exchange reactions, and forms an intramolecular disulfide at the active site in the process. Oxidized Trx relies on thioredoxin reductase (TrxR) for regeneration of its reduced state.65 The reduction of protein disulfides by the Trx/TrxR system is depicted in Scheme 1.3.  PSSP  Trx  SH SH  2PSH  Trx  FADH2  FADH2 HS  Se TrxR S HS  HS TrxR HS  SeH TrxR SH S  SeH SH  FAD  FADH2 S  S S  HS TrxR HS  NADP+  NADPH + H+  SeH SH  Scheme 1.3 Reduction of protein disulfide bond by thioredoxin/thioredoxin reductase system.65 PSSP is interprotein disulfide bond and PSH is the reduced protein  10  Another well known example of a biologically important intermolecular disulfide is that of glutathione disulfide (GSSG). The tripeptide glutathione (GSH) is the most abundant and important low-molecular weight thiol, and is ubiquitously distributed at millimolar concentrations in cells.  It has essential roles as an  antioxidant and intracellular redox buffer.66-68 GSH scavenges to remove ROS and during the detoxification of ROS produces GSSG, which can be reduced back to GSH by NADPH-dependent enzyme GSSG reductase.69  In addition, GSH is  involved in cell signaling processes and regulation of protein activity through modifications of the oxidation state of protein cysteines.  GSH can form mixed  disulfides with the protein Cys residues (PSHs) to generate S-glutathionylated proteins (PSSGs).70-72 Several mechanisms (Scheme 1.4) have been proposed for the formation of PSSG.  Among them is the thiol-disulfide exchange reaction  (Scheme 1.4, reaction A), where the thiol group of a Cys residue in protein reacts with the GSSG.66,73,74 The other proposed mechanisms (Scheme 1.4, reactions B-E) will be discussed in subsequent sections. Regardless of the route of S-glutathionylation, the process can be reversed by means of a reaction catalyzed by the thiol-disulfide oxidoreductase glutaredoxin (GRx).75,76 GRx has a conserved active site sequence Cys-Xaa1-Xaa2-Cys or CysXaa1-Xaa2-Ser and a GSH recognition site.77  GRx operates via a nucleophilic,  double displacement mechanism where the thiolate of GRx initiates a nucleophilic attack on the mixed disulfide of PSSG, leading to formation of a new disulfide between GRx and GSH and the release of the protein substrate in the reduced form. The mixed disulfide between GRx and GSH can be reduced by GSH through  11  another nucleophilic attack on the disulfide to form GSSG and reduced GRx as the final products (Scheme 1.5).78-80 Although other enzymes such as PDI, Srx and Trx can carry out this reversible process, GRx is preferentially used for this reaction.76,81,82  PSOSP + GSH (GSOSG) (PSH) PSNO (GSNO) E  GSH (PSH)  GSH (PSH)  D  PSOH (GSOH)  H2O C  NO  PSSG  O2  GSH  O2  GSSG A  PSSG  B  B  PSH  PS (GS ) + GSH (PSH)  PS + GS  Scheme 1.4  Formation of protein S-glutathionylation, A) Thiol-disulfide exchange, B) Cysteinyl radical, C) Cysteine sulfenic acid, D) Thiosulfinate, E) Snitrosylcysteine.  PSSG  GRx  PSH  GRx  S SH SSG  GSSG  GSH  SH  Scheme 1.5  Mechanism of GRx-catalyzed protein deglutathionylation.80  12  The existence of enzymes that selectively reverse S-glutathionylation (deglutathionylation) emphasizes the importance of S-glutathionylation in regulatory mechanisms.  S-glutathionylation regulates the structure and function of a wide  range of proteins, including enzymes, signaling molecules, transcription factors and ion channels.72  An example of S-glutathionylation is the regulation of actin  polymerization. Actin is a widely distributed cytoskeletal protein that is found to shuttle between monomeric and polymeric forms in cells.  In normal cellular  conditions, a portion of G-actin (globular actin, monomer) is S-glutathionylated at Cys374, which likely inhibits polymerization into F-actin (filamentous actin). In the presence of epidermal growth factor (EGF), deglutathionylation of Cys374 catalyzed by GRx leads to increase in polymerization of G-actin into filaments.83,84 This shows that cytoskeletal arrangements are regulated by the reversible S-glutathionylation of actin. At resting levels, S-glutathionylated proteins account for ~1% of the total. Oxidative stress leads to a significant increase in S-glutathionylation,85 which suggests that S-glutathionylation might also have a secondary role. In addition to its regulatory role, S-glutathionylation may serve as a means of storing GSH and of protection when the cell experiences oxidative/nitrosative stress, by preventing irreversible oxidation of critical Cys residues. This may often occur at the expense of a temporary loss of protein activity.66  This hypothesis is supported by S-  glutathionylation of γ-glutamyl transpeptidase, which appears to protect this membrane-bound enzyme from irreversible oxidation86 and the S-glutathionylation of α-ketoglutarate dehydrogenase that causes a reversible inactivation in response to  13  alterations in the mitochondrial GSH status.87 Although PSSG provides protection from overoxidation, these disulfides can be oxidized further to thiosulfinates (disulfide-S-dioxides, RS(O)SR) and thiosulfonates (disulfide-S-dioxides, RS(O)2SR) under highly oxidative conditions.88,89 disulfides can be reversed.  However, even such overoxidation of  In the presence of reducing thiols such as GSH,  thiosulfinates are readily converted to sulfenic acids (RSOH) which can then be reduced back to a thiol.90,91 Such a redox cascade is depicted in Scheme 1.6. The formation and fate of oxidized disulfides in vivo, however, still remain unclear.  RSH GSSG  GSH  RSSR O  Scheme 1.6  RSSG  GSH H2O  RSOH  GSH  Stepwise reduction of thiosulfinate.  1.4.2 Sulfenic Acids Cysteines can readily be oxidized to sulfenic acids (CysSOH), which are unstable and highly reactive. The formation of sulfenic acids is dependent on the Cys ionization state as thiolates are more nucleophilic than their protonated counterparts, thiols. CysSOH is formed by several different mechanisms as shown  14  in Scheme 1.7. CysSOH is formed predominately by the reaction of thiolate with H2O2 and other biological oxidants such as ONOO-, and HOCl.22,92-94 Four other less common reactions include the hydrolysis of protein disulfide bond through an enzyme-facilitated mechanism to generate one thiol and one sulfenic acid.95  A  second mechanism involves the oxidized disulfide, thiosulfinate, which can react with a thiol group to generate a new disulfide bond and CysSOH.89  Thirdly,  •  cysteinyl radicals can react with O2 or OH to form CysSOH.96 Finally, sulfenic acid is postulated to be formed by the hydrolysis of S-nitosothiols.97  RSNO RSSR  H2O  H2O  RSOSR  RSH  HOCl  RSH  HNO  H+ + Cl-  RSSR  RSH  RS + OH  RSOH NO2ONOO  RSH  H2O  RSOO  H2O2  RS  + O2  RSH  Scheme 1.7  Formation of cysteine sulfenic acid.  Once formed, sulfenic acids can generate other forms of reversibly or irreversibly modified cysteinyl groups. Sulfenic acids are notoriously difficult to study because they are highly unstable, thus evidence for the existence of stable CysSOH in  15  proteins are limited. Crystallographic evidence of CysSOH has been presented in NADH peroxidase.98  The stability of CysSOH is highly dependent on its  environment. The primary factor in its stability is the absence of proximal Cys group as CysSOH easily reacts with the vicinal thiol to form an intramolecular disulfide bond.  CysSOH can also interact with GSH to form S-glutathionylated protein.  Likewise, the sulfenated form of GSH (GSOH) can react with Cys in protein to form PSSG.99 When fixed in close proximity, the sulfur atom in CysSOH can also react with an amide nitrogen in the protein to form a cyclic sulfenamide (Scheme 1.8). This unusual modification was first observed directly in the crystal structure of the protein tyrosine phosphatase-1B100 and recently in the receptor protein tyrosine phosphatase-α.101 Sulfenamide can react with GSH to form the S-glutathionylated protein, therefore sulfenic acids play an important role in glutathionylation, which is a reversible process.  H N  R O  Scheme 1.8  O H R' N S OH  H N  R O  O N  R'  +  H2O  S  Formation of cyclic sulfenamide.  There are a number of proteins that are involved in sulfenic acid formation that play roles in enzyme catalysis, antioxidant defence, and cell signaling.  A  noteworthy example of sulfenic acids in enzyme catalysis and in antioxidant defence  16  comes from peroxiredoxins (Prx), a ubiquitous family of antioxidant enzymes that catalyze the reduction of H2O2 and other hydroperoxides. Prx enzymes possess one or two Cys residues in their active site (1-Cys and 2-Cys, respectively) and are divided into three classes: typical 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys Prx.102 All Prxs share the same basic catalytic mechanism in which the active site Cys residue is oxidized to CysSOH by the peroxide substrate. The recycling of the CysSOH back to a thiol is what distinguishes them into different classes (Scheme 1.9).  In 1-Cys Prx, the sulfenic acid likely reacts with a thiol-containing small  molecule forming a mixed disulfide bond which is then reduced by a second molecule of the reductant. The identity of the thiol-containing small molecule is not clear but GSH and lipoic acid have been proposed.102,103 In typical 2-Cys Prxs, the CysSOH from one subunit is attacked by the Cys residue of another subunit forming an intermolecular disulfide bond, which is then reduced by a disulfide oxidoreductase such as Trx.104,105  Finally, atypical 2-Cys Prxs have a similar  mechanism as typical 2-Cys but the Cys residue that is attacking the CysSOH comes within the same subunit forming an intramolecular disulfide bond.103,106 The capacity of Cys to cycle between its oxidized sulfenate and its fully reduced thiol form makes it a candidate as a regulatory mechanism in protein function, as has been proposed in protein tyrosine phosphatases (PTP). Reversible phosphorylation of proteins is an important regulatory mechanism. Within the cell, the level of phosphorylation at serine, threonine, and tyrosine residues are under the strict control of protein kinases (phosphorylation) and protein phosphatases (dephosphorylation). These enzymes are in turn regulated by diverse mechanisms.  17  PTPs catalyze the hydrolysis of phosphotyrosine from specific signal-transducing proteins.  PTPs along with protein tyrosine kinases (PTKs) are responsible for  maintaining a normal tyrosine phosphorylation status in vivo. Figure 1.4 shows the mechanism in which the activity of PTPs themselves may be regulated.  Upon  activation of a receptor PTK by interaction with epidermal growth factor (EGF) causes an increase in intracellular H2O2. This increase in H2O2 inhibits phosphatase activity of PTP due to the formation of CysSOH of the active site Cys residue.107,108 This reversible oxidation of Cys residue plays an important regulatory mechanism for downregulating PTP, thus enhancing tyrosine phosphorylation of proteins including receptor PTK. Dephosphorylation takes place after degradation of H2O2 and the subsequent reactivation of PTP by electron donors such as Trx.  1-Cys Prx ROOH  Prx  ROH  Prx-SOH  RSSR + H2O 2RSH  2-Cys Prx Prx  ROOH  ROH  Prx  SH SH  SOH SH  RSSR  Prx  S S  2RSH  H2O  Scheme 1.9  Mechanism of 1-Cys and 2-Cys peroxiredoxins.102  18  Figure 1.4  Mechanism of PTP activity regulated by H2O2. Y is tyrosine residue in protein and receptor. YP and PY are phosphorylated tyrosine residues.107  1.4.3 Sulfinic and Sulfonic Acids Sulfinic acid (CysSO2H) can be formed from sulfenic acid reacting with ROS such as H2O2 or via cysteinyl radical interactions with dioxygen (see Section 1.4.5). Unlike sulfenic acids, sulfinic acids do not undergo self-condensation reactions or react with thiols under physiological conditions.  Functional roles for sulfinic acid  have been identified in numerous proteins including D-amino acid oxidase (DAO),109 Parkinson’s disease protein DJ-1,110 and copper-zinc superoxide dismutase  19  (SOD1).111  In addition, CysSO2H acts as a ligand to the metal site in nitrile  hydratase (NHase), which catalyzes the transformation of nitriles to amides. In this protein, two Cys residues coordinated to the metal are modified to CysSO2H and CysSOH which is essential for catalytic activity.6,112 Sulfinic acid has received the most attention in the family of Prxs because of its reversibility by Sulfiredoxin (Srx).113,114 Unlike sulfenic acids, sulfinic acids cannot be reduced by major cellular reductants such as GSH and Trx. For this reason, it was generally believed that sulfinylation of proteins was irreversible until the relatively recent discovery of the ATP-dependent enzyme, Srx.115 However, Srx activity is limited to the reduction of CysSO2H in Prx proteins and does not provide a general route to desulfinylation. The mechanism of Srx-catalyzed sulfinic acid reduction in Prx proposed by Biteau et al.115 is shown in Scheme 1.10. The first step of the Srx reaction involves phosphorylation of sulfinic acid to form sulfinic acid phosphoryl ester because under physiological conditions, the hydroxyl group of CysSO2H (pKa ~ 2.1) is fully deprotonated and cannot serve as the leaving group. Therefore, phosphorylation of the sulfinic acid serves to improve its leaving group ability.  The sulfinic acid  phosphoryl ester is reductively cleaved by thiols such as GSH or Trx to produce a thiosulfinate.  The thiosulfinate is further reduced to the thiol form of Prx after  oxidizing three thiol equivalents. Formation and reversal of sulfinic acid in Prx indicates that Prx might play a role in cell signaling, a unique role for the eukaryotic Prxs. As mentioned above, Prx detoxifies H2O2 using the Cys/CysSOH redox cycle. In the presence of extreme  20  oxidative stress, the concentration of H2O2 exceeds the capacity of the enzyme and leads to CysSO2H formation. Once Prx is converted to CysSO2H, it is no longer enzymatically active, losing its antioxidant role and allowing for the build-up of H2O2.116  H2O2 starts the signaling cascade of various proteins117,118 and is  considered to function as an intracellular messenger at subtoxic concentrations. Therefore, Prx acts as a regulator of H2O2 mediated cell signaling.  ATP Prx S  ADP  OO  O O P OPrx S O O RSH HPO4-  H2O2  Prx S O  Prx SH  S R  RSH RSSR RSSR RSH H2O Prx S S R  RSH Prx S O  H  Scheme 1.10 Proposed mechanism of sulfiredoxin catalyzed sulfinic acid in peroxiredoxin.119  Sulfinic acids are stable intermediates, but are readily oxidized to sulfonic acid (CysSO3H) by ROS. Under intolerable oxidative stress, Prx loses its antioxidant 21  function and the cell signaling function due to formation of irreversible CysSO3H. The formation of sulfinic and sulfonic acids depends on the degree of oxidative stress.  Other than the sulfinic acid in Prx, both, sulfinic and sulfonic acids are  considered irreversibly oxidized forms of Cys; as such, they can impact protein function and homeostasis in many ways.  These highly oxidized species with a  negative charge distribution and steric requirements can impact protein structure and can inhibit enzyme activity of enzymes that require a thiolate for catalysis.  1.4.4 S-Nitrosylation S-nitrosylation is a ubiquitous modification of the cysteine thiol by nitric oxide •  •  (NO ). There is no evidence of a reaction of NO itself with Cys residues under •  physiological conditions, but the formation of NO quickly yields N2O3 and ONOO-, as mentioned earlier, both of which react with Cys residues to form S-nitrosylated proteins. Once formed, S-nitrosylated Cys can undergo reductive denitrosylation (catalyzed by Trx/TrxR)120 or form other reversibly oxidized Cys modifications. It can also promote disulfide formation with neighbouring thiols or react with GSH to form S-glutathionylated protein.  Transnitrosylation (transfer of NO group) by low  molecular weight S-nitrosothiols such as GSNO (S-nitrosoglutathione) has also been suggested as a possible mechanism for protein S-nitrosylation in a  manner  analogous to thiol/disulfide exchange (see Section 1.4.1).121,122 Reversibility of this Cys modification makes S-nitrosylation an ideal candidate for cellular regulatory mechanisms.  22  The physiological relevance of S-nitrosylation has been confirmed in mammalian plasma where S-nitrosoalbumin and S-nitrosoglutathione possess vasodilatory activity as well as the ability to inhibit platelet aggregation.123  S-  nitrosylation is implicated in cell signaling events within the cell. Critical signaling kinases such as extracellular signal regulated kinase (ERK), p38 and c-Jun NH2terminal kinase (JNK) are activated by nitrosylation.124 Ion channels such as Nmethyl-D-aspartate receptor channel complexes, Ca2+-activated K+ channels, cyclic nucleotide-gated channels, and cardiac Ca2+ release channels, are also postulated to be regulated by S-nitrosylation.125-128 S-nitrosylation is involved in host defence as well. For example, inactivation of the human immunodeficiency virus (HIV)-1 protease can be achieved due to Cys nitrosylation.  HIV-1 protease action is  modulated by the redox equilibrium of Cys residues at position 67 and 95 and the nitrosylation of these Cys residues inactivates the protease activity which is essential for the life cycle of HIV.129  1.4.5 Cysteinyl Radical There are several routes for the formation of cysteinyl radicals in biology. The general mechanism of GSH antioxidant activity is known to occur through abstraction of the S-bound H atom by carbon-centred radicals with concomitant formation of the cysteine-based radical of GSH.130 Reaction of Cys with ROS and •  RNS such as OH and ONOO- also generates cysteinyl radicals.131-133 Cysteinyl radicals also occur in important enzymes such as the ribonucleotide reductase (RNR) superfamily, pyruvate formate lyase (PFL), and benzylsuccinate synthase  23  (BSS). Cysteinyl radicals in these enzymes are formed by one of two mechansims: (i) long-range one-electron transfer from the Cys or (ii) short range H-atom donation from the Cys.91 Long-range one-electron transfer occurs in aerobic Fe-dependent (class I) RNR from E.coli, which generates a cysteinyl radical by electron transfer from Cys to a remote tyrosyl radical.134 Active site cysteinyl radical formation in anaerobic formate-dependent (class III) RNR from bacteriophage T4 and in anaerobic PFL from E.coli proceeds through a short-range hydrogen-atom donation from Cys residue to a glycyl radical.135  Furthermore, cysteinyl radicals can be  generated in the presence of iron or copper ions.136,137 Once formed, a cysteinyl radical can participate in a number of different reactions, (Scheme 1.11).  They can be reduced back to Cys through H-atom  abstraction. In RNR the hydrogen atom is abstracted either from external dithiols (for class I) or formate (for class III), which are ultimately oxidized to disulfides or CO2, respectively.138 However, cysteinyl radicals can also abstract hydrogen from nearby amino acids, which may be a starting point for protein aggregation and/or fragmentation that lead to irreversible protein damage.  In  addition, cysteinyl •  radicals can react with thiolates or thiols to form disulfide radical anions (RSSR -), which in turn can react with oxygen to form a disulfide and superoxide that can be removed by SOD.139  This is a possible mechanism for the formation of S•  •  glutathionylated protein in which cysteinyl radical (PS or GS ) react with GSH or •  PSH, respectively. Cysteinyl radical could alternatively be intercepted by OH or NO  •  to yield sulfenic acid or S-nitrosothiol, respectively.96,140  24  RSNO  RSO  O2  H2O  RSOH OH  NO RS  RSSR  RSH  RS  HO2  O2  RSOO  RSOOH RSH  O2  H2O2 RSOH  RS  H2O2  RS  H2O  O2 RSSR  O R S O  RSH  HO2  O2  RSH  RS  O R S OH  H2O2 H2O  O R S O  O R S OO O  O2  H2O  HO2  O R S OH O  Scheme 1.11 Reactions of cysteinyl radicals.  Cysteinyl radicals can also react with oxygen to form thiyl peroxyl radical (RSOO ).  •  RSOO can further react to form a number of other highly reactive  •  species.  RSOO can isomerize to the fully sulfur-centered, thermodynamically  •  •  favoured sulfonyl radical (RSO2 ).  RSO2• may subsequently react with another •  oxygen and give rise to sulfonyl peroxyl radical (RSO2OO ).141 The highly reactive •  RSO2OO intermediate converts ultimately to RSO3H by reaction with water.142 •  •  RSOO can also oxidize Cys residues yielding a sulfinyl radical (RSO ) and sulfinic •  •  •  •  acid.143 All of these radicals species (RSOO , RSO2 , RSO , RSO2OO ) exhibit nonspecific oxidizing properties and therefore can be very damaging to cells. Moreover,  25  •  RSOO is seen to be the precursor of sulfenic, sulfinic and sulfonic acids and disulfide.144 Thiyl hydroperoxides (RSOOH) are possibly present as an intermediate in these processes.  •  RSOOH is generated from RSOO via H-abstraction from •  another thiol group or via reaction with HO2 .145 RSOOH can interact with water to yield H2O2 and RSOH or it may react with another thiol molecule to form a disulfide. RSOOH can generate RSO2H via isomerization which can then react with H2O2 to yield the highly oxidized Cys, RSO3H.146 Cysteinyl radicals are considered to be highly reactive species that act as intermediates in the reversible or irreversible formation of other Cys oxidation products. Cys radical generation is important for several biological mechanisms, such as the enzymatic functioning of RNR and PFL, cell signaling (as intermediates of S-nitrosylated proteins), PSSG and sulfenic acid, and antioxidant defence mechanism provided by GSH. It is worth mentioning that the antioxidant defence mechanism provided by Cys radicals is counterbalanced by its ability to abstract hydrogen, which can damage other biomolecules such as amino acids, carbohydrates, and lipids.147-149 Therefore, the Cys radical is considered a doubleedge sword in these biological redox processes. The amino acid Cys has been regarded as a sink for oxidation processes in peptides and proteins. As discussed above, there are various oxidation products of Cys and the mechanism of Cys oxidation is very complex. The complexity of Cys oxidation is illustrated in Figure 1.5 to give an overall view of each Cys oxidation product and how they interplay with each other.  26  Figure 1.5  Formation of cysteine oxidation products in proteins and peptides. Irreversible oxidation products are framed with dashed lines. The dashed arrow represents one exception where occurs in Prx.  27  1.5  Methionine Oxidation Methionine is susceptible to oxidation as well but, unlike Cys, it has fewer  oxidized products (Figure 1.6). Met oxidation also involves reversible as well as irreversible oxidation processes.  R  H N  O R'  S  Methionine (-2)  Figure 1.6  R  H N  O R'  H N  R  S Methionine Cation Radical (-1)  O R'  S  O  Methionine Sulfoxide (0)  R  H N  O R' S O O  Methionine Sulfone (+2)  Common oxidative modifications of methionine in proteins that have been observed or proposed to occur in vivo. (Formal oxidation states for the sulfur atom in these species are given in parentheses.)  1.5.1 Formation and Role of Sulfoxide Methionine is quite easily oxidized to methionine sulfoxide (MetSO).  The  biological oxidants that mediate the formation of MetSO are superoxide, hydrogen peroxide, hydroxyl radicals, singlet oxygen, hypochlorous acid, and chloramines.19,22 Met oxidation leads to a mixture of both the S-and R-stereoisomers of MetSO. MetSO can be reduced back to Met by methionine sulfoxide reductases (Msr) in the presence of Trx. Msr are small cytosolic enzymes found in a variety of organisms ranging from bacteria to mammals.150-153 There are two classes of Msr, depending  28  on the stereochemistry at the sulfur center. MsrA is specific for the reduction of (S)MetSO whereas MsrB is for (R)-MetSO.154,155 Two possible reaction mechanisms for the Msr have been proposed; proceeding either through a series of thiol-disulfide exchange reaction steps or through a sulfenic acid intermediate formation. Scheme 1.12 shows the mechanism of bovine MsrA which undergoes the thiol-disulfide exchange reaction. The mechanism is initiated by the nucleophilic attack on the sulfoxide sulfur atom by the thiolate anion of Cys72 which leads to the formation of a thiosulfinate intermediate. Subsequent attack by the thiolate anion of Cys218 and a proton transfer to the sulfoxide oxygen leads to loss of water and formation of Met along with formation of a disulfide bond between Cys72 and Cys218.  The initial  (reduced) state of the active site is then restored with a thiol-disulfide exchange between Cys227 and Cys218 followed by the reduction of this disulfide bond with Trx.156 In the case of E. coli MsrA (Scheme 1.13), the initial intermediate that is formed by Cys51 attack on the sulfur atom of sulfoxide rearranges to a sulfenic acid. During this process Met is released with the oxygen atom transfer from Met to the Cys51. Cys198 then attacks Cys51 to form a disulfide bond and release of water. The active site is then restored by thiol-disulfide exchange, first by Cys206 then by Trx.157  29  Scheme 1.12 Proposed mechanism of bovine methionine sulfoxide reductase.156  The reversibility of MetSO back to Met suggests that MetSO has a biochemical and physiological role.  Met oxidation to MetSO can serve as an  antioxidant.158 Surface Met residues in proteins act as protection against oxidation of other residues that are vital for activity.  This protective role is observed in  glutamine synthetase where 8 of the 16 Met residues are oxidized to MetSO without significantly affecting the enzyme activity. The Met residues near active sites can prevent autoxidation of other amino acids by substrates, products, or cofactors.18 A further example of Met serving as an antioxidant comes from α-2-macroglobulin, a physiologically important proteinase inhibitor, which acts at sites of inflammation  30  where ROS and RNS are in relatively high concentration.  In such oxidizing  environments, Met residues in α-2-macroglobulin get oxidized to MetSO without the loss of antiproteinase activity.  However, prolonged exposure to this oxidizing  environment causes a single tryptophan (Trp) residue to be oxidized with concomitant loss of antiproteinase activity. This suggests that Met residues serve to protect the critical Trp from damage.159,160  Scheme 1.13 Proposed mechanism for E.coli methionine sulfoxide reductase.157  31  The reversibility of MetSO formation also suggests that it may play a role in cell regulation. Evidence for this comes from the Ca2+ binding protein calmodulin (CaM). CaM is involved in many cellular processes as it controls the function of a number of enzymes, ion channels, pumps, and other signaling proteins that depend on Ca2+. Upon C-terminal Met oxidation to MetSO in CaM, the protein loses its conformational stability and therefore loses its function.161 Msr can restore CaM function by reducing MetSO back to Met.162 This cyclic oxidation and reduction of the specific Met in CaM constitutes a regulatory system by which the activity of numerous cellular signaling systems can be controlled.  1.5.2 Formation and Role of Sulfone Methionine sulfoxides can be further oxidized by H2O2 to methionine sulfones (MetSO2), which are the most oxidized form of Met; no biologically-relevant reduction of MetSO2 has been observed. The biological role of MetSO2 is less apparent. Evidence of MetSO2 is usually observed in pathophysiological states.110,163 However in a unique example, MetSO2 is shown to act as a ligand and portray a beneficial role. In catalase of Proteus mirablis PR, a peroxide resistant mutant of P. mirabilis which contains a heme group that interacts with a MetSO2. Catalase, an enzyme found in almost all aerobic organisms catalyzes the dismutation of H2O2.  The  oxidation of Met53 could result from the action of H2O2 and the presence of MetSO2 does not significantly affect the activity of the catalase. It has been proposed that this formation of MetSO2 could produce some steric hindrance impairing the accessibility of large substrates or inhibitors to the iron containing active site. P.  32  mirabilis PR catalase is less sensitive to aminotriazole, a specific inhibitor of catalase known to bind covalently to the essential distal iron coordinated by histidines.164  1.5.3 Methionine Radical Cation •  Formation of the methionine radical cation (MetS +) has been suggested, but concrete evidence of such formation in vivo is still lacking. Like the cysteinyl radical, •  MetS + may act as an intermediate in the formation of MetSO.  •  MetS + is proposed  •  to be formed via metal-induced oxidation and by ROS such as OH.165-167 •  One example where the biological relevance of MetS + has been proposed is in the chemistry of amyloid-β (Aβ) peptide and its relevance to Alzheimer’s disease (AD).  AD is a progressive neurodegenerative disorder with two characteristic  features, extracellular deposits of amyloid-β (Aβ) peptide and neurofibrillary tangles.168-170 Evidence suggests that the aggregation of Aβ is caused by abnormal interactions with metal ions such as Zn2+, Cu2+ and Fe3+. Aβ is a relatively small 40 to 42 amino acid long membrane protein with metal binding sites in the N-terminal region.171 Binding of redox-active metal ions (i.e., Cu2+ or Fe3+) is believed to result in the oxidation of Met at position 35. Although, thermodynamically the one-electron transfer from Met35 to Cu2+ is not favoured, it is enhanced by neighbouring isoleucine (Ile) at position 31. Pulse radiolysis studies have shown that sulfide radical cations can be stabilized through complexation with Lewis bases.172  Also, one-electron  oxidation of the model compound N-acetyl-L-methionine amide has shown that the  33  thioether radical cation is stabilized through complexation with the amide bond forming a cyclic structure (Scheme 1.14).167  Thus, it is hypothesized that one-  electron oxidation of Met35 in Aβ is stabilized through complexation with carbonyl oxygen of C-terminal of Ile31 forming a S-O bonded cyclic radical.165,173  H N  H3C  O NH2  O  -e  H N  H3C O  S  NH2 O S  CH3  Scheme 1.14 One electron oxidation of N-acetyl-L-methionine amide.  •  The formation of MetS + is hypothesized as a possible explanation of how a membrane bound peptide is found to be in the extracellular space of AD individuals. Structural studies indicate that residues 28-36 of Aβ form a helix, which is embedded into the membrane. Oxidation of Met35 disrupts the helical conformation and causes Aβ to be dislodged from the membrane forming insoluble aggregates.174 Evidence •  of MetSO in Aβ plaques have been observed175 which suggest that perhaps MetS + is the intermediate in the metal-induced oxidation of MetSO. AD is not the only unique case where Met oxidation is implicated. Cys and Met oxidation are thought to be involved directly or indirectly in various disease states.  34  1.6  Implications of Cys and Met Oxidation in Diseases The irreversible oxidation of Cys and Met in proteins and peptides may lead  to loss of function and therefore to pathophysiological states.  Similarly poor  regulation of reversible Cys and Met oxidation would have similar consequences. When ROS and RNS are produced in excessive amounts, oxidative and nitrosative stress occurs, respectively. Cells try to cope with the oxidative and nitrosative stress through sophisticated antioxidant defence mechanisms, including enzymatic and non-enzymatic processes, and through specific repair mechanisms discussed previously. Glutathione, one of the major antioxidants involved in detoxifying ROS and RNS, gets oxidized to GSSG.  GSSG accumulates in stressed cells and an  imbalance in the ratio of GSH/GSSG occurs if cellular processes cannot effectively reduce GSSG back to GSH; the GSH/GSSG ratio has therefore been used as an indicator of oxidative/nitrosative stress in an organism.176 A high concentration of GSSG may damage many enzymes oxidatively. GSH is also a cofactor for several detoxifying enzymes against oxidative stress such as glutathione peroxidase and glutathionetransferase. Thus, the unavailability of the reduced GSH affects these enzymes  and  causes  more  accumulation  of  ROS  and  RNS.  Under  oxidative/nitrosative conditions, an increase in S-glutathionylated proteins occurs to protect the Cys residues in the proteins from irreversible oxidation. A significant increase in PSSGs have been found in diseases such as cancer, HIV, and diabetes mellitus.177-179 There is also evidence of low GSH levels in lymphocytes of HIV patients.180 HIV-infected cells have decreased ability to dethiolate PSSGs178, as  35  mentioned earlier, dethiolation is dependent on free GSH. Thus, S-glutathionylated proteins have been investigated as possible biomarkers of oxidative stress in correlation with disease. Since the blood concentration of PSSGs may reflect the state of oxidative stress status even in inaccessible tissues, hemoglobin is used as a biomarker for some human diseases.181 For example, glutathionylated hemoglobin is increased in patients suffering from type I and type II diabetes, Friedreich’s ataxia, hyperlipidemia, and uremia.182-184 Similarly, alterations in the balance between thiols and their S-nitrosylated forms are believed to be indicative of disease states. Significant increase in Snitrosylated proteins has been observed in joints of arthritis patients due to the increased level of NO production.185  In diabetes mellitus, elevated levels of  nitrosylated hemoglobin in red blood cells have been observed, which impairs the vascular relaxing activity.186 Human serum albumin (HSA), the most abundant thiolcontaining molecule in plasma, is endogenously S-nitrosylated.187 Accumulation of nitrosylated HSA in pre-eclamptic women contributes to hypertension because nitrosylated HSA is a weak vasodilator.188,189 Antioxidant enzymes such as SOD, Prx and GSH peroxidase can also be affected by ROS/RNS as well.116,190,191  For example, copper-zinc superoxide  dismutase (SOD1) plays a protective role against oxidative stress, however it becomes a major target of oxidative damage itself leading to pathogenicity in various neurodegenerative diseases, including familial amyotrophic lateral sclerosis.190 SOD1 in humans has a reactive Cys residue, Cys111, at the surface of the SOD1, which is selectively oxidized to the Cys sulfinic acid and to Cys sulfonic acid.192  36  Imbalance between ROS/RNS production and antioxidant enzymes causes an increase in Cys and Met oxidation. This imbalance has been shown to play a role in the pathogenesis of a number of diseases.193-195 In addition to overwhelming the antioxidant defence mechanism under extreme oxidative/nitrosative stress, repair mechanisms can also be affected and as a result can increase the oxidation of Cys and Met. Several studies indicate that the level of Msr declines with age-related diseases such as Alzheimer’s disease, Parkinson’s disease and cataractous lenses.163,196-198 This decrease in Msr causes accumulation of MetSO which has the potential to be oxidized to the irreversible MetSO2.  There is also an increase of MetSO in human skin collagen and  erythrocytes199-201 These various studies are consistent with a generalized agedependent increase in MetSO content in proteins. Finally, an increase in ROS and RNS can oxidize Cys and Met to the irreversible forms, which can lead to loss of protein function and hence to the formation of disease.  Oxidative damage is thought to be a major factor in the  development of age-related cataracts, a leading cause of blindness in the world.202 Characteristics of age-related cataracts include extensive oxidation, cross-linking, and insolubilization of lens proteins.203 α-Crystallin is one of the most abundant lens proteins that are found extensively in cataract protein aggregates. Its function is to maintain proper refractive index and to function as a molecular chaperone, helping to prevent formation of large light-scattering aggregates.204,205 Oxidation affects its chaperone activity which suggests that oxidation may have important consequences for protein aggregation in the lens.206 Among the amino acids that are oxidized, Cys  37  and Met are found to be in oxidized forms to a large extent. Cys is oxidized to sulfinic and sulfonic acid and Met is oxidized to the sulfoxide and the sulfone forms.163 As mentioned in the previous section, Met oxidation is implicated in AD but Met oxidation is also implicated in other neurodegenerative diseases such as Parkinson’s disease (PD). Like other neurodegenerative disease, PD also has the characteristic feature of protein fibril (amyloid) deposits.  α-Synuclein is a small,  natively unfolded and soluble protein that is found in cytosol and is associated with presynaptic vesicles.207 The normal function of this protein is not known, but it is found in the aggregates characteristic for several neurological diseases and therefore thought to be a key player in the pathogenesis. Numerous observations suggest that oxidative stress may be associated with PD, and the aggregation of αsynuclein is a critical component in the etiology of PD. The lack of Trp and Cys residues leaves few alternatives for the oxidation of α-synuclein and thus Met is found to be oxidized to MetSO.  Formation of MetSO increases the degree of  unfolding of this protein.208 However, the relationship between protein oxidation, protein aggregation and neurodegeneration in PD is still unclear.  38  CHAPTER 2: X-RAY ABSORPTION SPECTROSCOPY  2.1  Background and Theory X-ray absorption spectroscopy (XAS) results from the absorption of a high  energy X-ray by an atom in a sample. As X-rays with increasing energy are passed through the sample, absorption will occur at a specific energy (threshold) through the process of exciting a core electron into empty or partially-empty bound electronic states or can be ionized to the continuum of unbound states. The ionization to the continuum is a characteristic feature of XAS termed “edge”.209,210  Thus, XAS  measures the absorption, μ, by a sample as function of photon energy, E which is usually expressed in units of electron-Volts (eV). The shell and the subshell from which the electron is ejected gives rise to the edge’s name. The principle quantum number is represented as an uppercase letter and the specific absorption line as a subscript number. For example 1s = K, 2s = L1, 2p1/2 = L2, 2p3/2 = L3, 3s = M1 and so on.211,212 XAS is an element specific technique since the energies of the edges (ionization energies) are generally well separated from their nearest neighbours. For example, the S K-edge occurs at approximately 2472 eV, whereas the P and Cl Kedges are at 2146 eV and 2822 eV, respectively.211 The core electron excitation depends not only on the element but also its chemical environment, so XAS shows also chemical specificity. Therefore, XAS is sensitive to oxidation state, coordination number, and geometry. XAS can be used for almost any element in the periodic  39  table. Importantly, crystallinity is not a requirement for XAS measurements, thus non-crystalline and highly disordered materials can be probed. Its sensitivity allows the XAS investigation of dilute solutions as well. A typical XAS spectrum is commonly divided into two main regions: the X-ray absorption near-edge structure (XANES) and the extended X-ray absorption fine structure (EXAFS) (Figure 2.1).  Together, XANES and EXAFS can provide  complementary information, yet the analysis techniques in these two regions are completely different. The XANES region consists of the pre-edge and the edge. The EXAFS region typically starts at ~40 eV above the edge and can extend to several hundred electron volts above the edge.213  XANES  Figure 2.1  EXAFS  XAS spectrum depicting XANES and EXAFS regions.  40  2.1.1 X-Ray Absorption Near-Edge Structure (XANES) The XANES region, as mentioned, consists of bound-state transitions (preedge) and ionization (edge).  The pre-edge transitions normally follow dipole  selection rules, i.e., ∆l = ±1, but electric quadrupole-allowed transitions can also be visible, particularly at metal edges. For the first-row transition metal K-edge XANES, dipole allowed 4p ←1s and electric quadrupole allowed but dipole forbidden 3d←1s transitions may be observed.  The quadrupolar peaks are observed below the  absorption threshold (edge). These pre-edge transitions are due to empty valence d-orbitals of the metal which participate in bonding and they are split in energy depending on the ligand field around the absorbing metal.214 The intensity of preedge peaks is generally weak, but can provide information on the coordination environment and charge of transition metal atoms. The dipole allowed bound-state transitions usually dominate the XANES region.  The edge jump, a step-like  increase, is a unique XAS feature that corresponds to the ionization of the electron to the continuum. Figure 2.2 shows the transitions observed in XANES region and the spectral features. The XANES region is sensitive to the oxidation state and speciation of the element of interest, and consequently is often used to determine the oxidation state and coordination environment of materials.  XANES is capable of distinguishing  species of similar formal oxidation states but different coordination. XANES spectra are commonly compared to standards to determine which species are present in an unknown sample.  Sulfur K-edge XAS shows particularly rich pre-edge spectra  corresponding to dipole allowed transitions that involve excitation of a 1s electron to 41  antibonding molecular orbitals, which are formed with significant contribution from the sulfur 3p-orbitals.215 S K-edge spectrum exhibits sharp linewidths and a large chemical shift range over its range of oxidation states216 as shown in Figure 2.3. As the formal oxidation state of the sulfur increases, the energy of the dipole allowed major peak of the pre-edge spectrum increases as well. These pre-edge transitions provide a sensitive probe of electronic structure and hence of chemical form.  Figure 2.2  XANES transitions and spectral features.  42  Figure 2.3  Sulfur K-edge spectra of series of oxidized sulfur species.  43  2.1.2 Extended X-Ray Absorption Fine Structure (EXAFS) The EXAFS region refers to the sinusoidal waves of the X-ray absorption, μ, observed at energies above the edge as a function of photon energy, E.  For  isolated atoms, μ decreases monotonically as a function of E beyond the edge. For atoms either in a molecule or embedded in solid, liquid or matrix, μ displays a fine structure caused by backscattering of the ejected photoelectron from neighbouring atoms.217  The ejected photoelectron wave propagates out of the central atom  (absorber). This propagating electron can be scattered by the neighbouring atoms (scatterers). This scattered wave can either interact constructively or destructively with the outgoing spherical wave resulting in a “peak” or “trough”, respectively in the EXAFS region, Figure 2.4.  Figure 2.4  The constructive and destructive interference of photoelectron waves that give rise to EXAFS.  44  Since the oscillation of the X-ray absorption is a direct consequence of the interactions between the photoabsorbing atom and its surrounding environment, EXAFS provides structural information. EXAFS allows quantitative determination of the scatterer’s identity (Z ± 1), the number of each type of scatters (N ± 25%), and the interatomic distance between the photoabsorber and scatterers (R ± 0.02 Å).210 Because EXAFS results from the wave behaviour of the photoelectron, it is common to convert the X-ray energy to k, the wave number of the photoelectron, which is defined as: Equation 2.1 Photoelectron wavenumber, k  k=  2m( E − E0 ) 2  Where: m = mass of the photoelectron  = Plank’s constant, h, divided by 2π E = incident photon energy E0= ionization energy of the core electron  For a single absorber-scatterer path the EXAFS equation can be represented as shown in Equation 2.2. This equation can be broken down into two components, the amplitude and the phase functions. The amplitude of the EXAFS can be dampened by two exponential terms. The first exponential term is related to the mean-free path of the scattered photoelectron and accounts for inelastic loss. The latter is called the Debye-Waller factor, which accounts for the thermal vibrations and static disorder of the system.210  Lower experimental temperatures reduce the thermal vibration,  resulting in larger amplitudes and improved signal to noise ratios. The EXAFS for any absorber-scatterer pair is represented as a damped sine wave with the  45  amplitude, frequency and phase shift characteristic of the atoms involved. The total EXAFS is the sum of the individual sine waves describing each absorber-scatterer pair interaction.  There are two types of parameters present in Equation 2.2:  parameters such as S02 and λ (k ) that are necessary to account for the scattering process and parameters such as Ni , Ri , and σ i2 that bear structural information. The structural parameters are relevant in understanding the metal sites in bioinorganic systems. Table 2.1 summarizes the structural information that can be extracted from an EXAFS region.  Equation 2.2 EXAFS single-scattering equation  N i Ai (k ) exp ( −2 Ri / λ ( k ) ) exp ( −2σ i2 k 2 ) sin [ 2kRi + φi (k ) ] 2  kRi i  phase function amplitude function  χ (k= ) S02 ∑  Where: Ni = number of atom i scatterers Ri = absorber-backscatterer distance for atom i σi2 = mean-square deviation in Ri (part of the Debye-Waller factor) S02 = total amplitude reduction factor; accounts for electronic relaxation λ(k) = photoelectron mean free path function Ai(k) = backscattering amplitude function (including amplitude reduction function, Si(k) and EXAFS amplitude function, fi(k)); Ai(k) = Si(k)fi(k) φi(k) = total EXAFS phase shift function (including scatterer, ψi(k), and absorber, δa(k), phase shift functions); φi(k) = ψi(k) + δa(k) k = photoelectron wavenumber  46  Table 2.1  Summary of structural information extracted from EXAFS. Structural Information  From sine wave  Distance between absorber and scatterer (Ri)  Frequency  Coordination number (Ni)  Amplitude  Scatterer identification (Z)  Phase shift and amplitude  Debye-Waller factor (σi2)  Damping effect  As mentioned above, Equation 2.2 is only applicable to a single-scattering model where each photoelectron is backscattered from one atom.  However, in  addition to single-scattering, multiple-scattering can also occur in which the photoelectron is scattered by two or more atoms before returning to the absorber. The net effect of scattering by multiple atoms is that the amplitude and phase functions are modified. The strength of a multiple-scattering path depends on the angle between the central atom and the scattering atoms (scattering angle). In most cases, three-body multiple scattering interactions contribute to the EXAFS region (Figure 2.5). For such interactions to have significant intensity, the scattering angle has to be ≥150o. Multiple-scattering is also important for rigid structures such as histidine or tyrosine residues.209  A combination of all the single- and multiple-  scattering processes results in the final EXAFS region. There are several EXAFS fitting programs which incorporate single- and multiple-scattering theory.  47  Figure 2.5  2.2  Scattering pathways in a three-body configuration consisting of absorber (A) and scatterer (S). (I) is the single-scattering pathway and (II) and (III) are possible multiple-scattering pathways.  Experimental Set-up Since the excitation of electrons requires high energies, this technique  demands high energy and tunable X-ray sources.  As a result, it is done at  synchrotron radiation facilities. All the XAS data for this thesis were collected at the Stanford Synchrotron Radiation Lightsource (SSRL). Synchrotron radiation is the electromagnetic radiation produced by the acceleration of electrons traveling at a velocity close to the speed of light in a magnetic field. Electrons are accelerated in a booster ring to relativistic speeds and directed to the storage ring. Within the storage ring, large dipole magnets guide the electrons around a “curved” orbit. The storage ring in reality is not circular but a many sided shape with straight sections.  In these straight sections, insertion  devices such as undulators and wigglers, containing a specialised set of magnets are placed to intensify the beam.218,219 X-ray radiation is emitted tangentially to the electron path in the magnetic fields of the ring. A schematic of the X-ray emission is  48  shown in Figure 2.6. The emitted photons contain electromagnetic radiation ranges from hard X-ray to infrared wavelengths, and can be tuned to a desired wavelength using various optical devices and filters.218  Figure 2.6  Schematic of a synchrotron  The research presented in this thesis was performed at two different beamlines at SSRL. The sulfur K-edge XAS data were collected from beamline 6-2 and the metal K-edge XAS data (hard X-ray) were collected from beamline 7-3. The basic set-up of these beamlines differs slightly.  A simplified schematic of the  beamlines are shown in Figure 2.7.  49  Figure 2.7  XAS experimental set-up  The size of the beam is defined vertically and horizontally using slits before passing through a double crystal monochromator, which is used for energy selection. Two parallel crystals diffract the beam according to Bragg’s law: Equation 2.3 Bragg's Law  nλ = 2d sin θ where λ, n, and d are the wavelength of the diffracted X-rays, the harmonic order, and the lattice spacing at that plane at a Bragg angle θ from the beam direction. The X-ray wavelength is controlled by a computer system, which rotates the monochromator so that θ changes at a constant rate per unit time.  Thus  measurements over the entire energy region of interest can be taken. The monochromatized beam is further defined in size using a set of vertical and horizontal slits before it enters the experimental hutch. For beamline 6-2, the beam enters a He-filled ionization chamber (I0), which is used to measure the intensity of the incident beam. The I0 chamber in beamline 7-3 is filled with N2 gas.  50  The beam then passes through the sample compartment and the transmitted light (I1) is detected in a second ion chamber providing the transmission data I1/I0. For dilute solution samples, the I1 signal is usually very weak and fluorescence detection mode is used.  At beamline 6-2, the S K-edge XAS measurements are only  measured using fluorescence mode. fluorescence from the sample.  A Lytle detector is used to collect the  In the hard X-ray set-up (beamline 7-3), a Ge  detector is used which has an array of germanium crystals that simultaneously collect fluorescence data, which are averaged together to reduce noise. In either set-up the fluorescence detector is placed at 90o to the incident beam and the sample is oriented at a 45o angle from both the incident beam and to the detector. The spectrum is obtained as FF/I0. All the data in this thesis were collected in fluorescence mode. In the case of hard X-ray measurements, internal calibration of the incident beam energy is possible by measuring the absorption of a reference sample using a third ionization chamber I2, providing the reference transmission data I2/I1.  In  contrast, beamline 6-2 does not provide internal calibration. An external calibrant needs to be run before and after the sample for proper energy calibration.  In  beamline 7-3, the sample is located in a vacuum chamber with a helium cryostat that maintains a constant low temperature (usually < 20 K), which helps prevent or reduce photoreduction of sensitive samples as well as minimise the disorder. In beamline 6-2, samples can be maintained from room temperature to < 20 K with a helium cryostat, depending on the type of experiment.  51  2.3  Data Reduction and Analysis XAS data reduction and analysis requires a number of steps and a variety of  computer programs are used.  2.3.1 Sulfur K-edge XANES SixPACK software was used for S K-edge data reduction.220  The raw  fluorescence data were inspected to eliminate beam or sample induced abnormalities. To ensure that the data were reproducible, two or more sweeps * were run for each sample. The sweeps of a run were averaged together and the energy position was calibrated using reference data.  Pre and post-data energy  calibration scans were compared, and the energy scale of the data was adjusted by taking the first pre-edge feature of sodium thiosulfate (Na2S2O3) and setting this to 2472.02 eV. A smooth second-order polynomial background was fitted for the preedge region and subtracted from the entire spectrum. The intensity of the edge jump is concentration dependent, thus normalization is required to enable comparisons between different samples.  Normalization of the data was  accomplished by fitting a flattened second-order polynomial to the post-edge region and normalizing to an edge jump of 1.0.  Energy positions of features in the  *  One sweep corresponds to the energy scan from low to high energy for the relevant region, producing an XAS spectrum.  52  normalized spectrum were obtained from the minima of the second derivative spectrum of the data.  2.3.2 Metal K-edge XANES and EXAFS ATHENA software221 was used for Cu and Fe K-edge XANES data reduction. The data processing is similar to the procedures used for analysis of S K-edge. For energy calibration, the lowest-energy maximum of the first derivative of the reference spectra was assigned, for Cu K-edge was set to 8979 eV and for Fe Kedge was set to 7112 eV. The averaged energy calibrated data was used for EXAFS processing but the steps required for this is different from XANES. The EXAFS region has an oscillatory fine structure described by χ (E ).  To  process the data in this region, a smooth pre-edge function was first subtracted, then χ (E ) is extracted from the spectra by fitting a spline function, μ0(E), above the edge. μ0(E) corresponds to the absorption of an isolated atom. In addition, EXAFS data was normalized to the edge jump of the smooth atomic-like background, ∆μ0 (Equation 2.4, Figure 2.8A) Equation 2.4 Oscillatory fine structure, χ (E )  χ (E ) =  µ (E ) − µ0 (E ) ∆µ0  Once the final χ (E ) function has been extracted, the oscillations can be viewed in k-space (Å-1), which is usually weighted as k3 to emphasize the high k region (Figure 2.8B). The Fourier transformation is used to convert the data from k-  53  space to R-space (Å), a more conceptually useful depiction (Figure 2.8C).  The  Fourier transformation gives peaks at the distances between the absorber atom and its neighbours, although slightly shifted from the true value of R.  Figure 2.8  EXAFS data processing: A) spline function, B) k3-space and C) R-space plots.  In order to get the structural information from EXAFS, experimental data need to be fit to a hypothetical structure, either theoretically calculated or built up from a known structure. Fitting can be performed in k-space or in R-space. ARTEMIS program,221 an interface to IFFEFIT222 is used for EXAFS fitting. Parameters such as amplitude, phase and mean-free path are calculated by FEFF6223 and are used 54  for iterative data fitting.223  The hypothetical model is also refined by varying  structural parameters to minimize its difference with the experimental spectrum.209 Table 2.2 lists the variables that are calculated and those that are refined during the fitting process. The quality of the fit is determined by the goodness-of-fit parameter, R-factor (Equation 2.5), which is automatically calculated by ARTEMIS. The Rfactor is essentially the fractional misfit. Values under 0.02 are widely considered to indicate a close match, however, for dilute biological samples values up to 0.2 are considered acceptable. Equation 2.5 R-factor N  R=  N N −n  ∑ χ (k )  2  i theory  − χ (k i )exp k ip  i  N  ∑ χ (k ) i  2  k ip  i  Where: N = number of experimental data points n = number of fitting parameters (variables) χ (ki )theory = theoretical EXAFS signals  χ (ki )exp = experimental EXAFS signals k ip = k weighting factor (p = 3)  Table 2.2  Calculated and refined parameters in the EXAFS fitting process. Calculated Parameters  Refined Parameters  Backscattering amplitude function  Ai(k)  Interatomic distances  Ri  Total phase shift function  φi(k)  Number of scatterers  Ni  Mean free path function  λ(k)  Disorder parameters  σi2  Amplitude reduction factor  S02  Threshold energy  E0  55  CHAPTER 3: SYNTHESIS AND CHARACTERIZATION OF CYSTEINYL RADICALS IN PSEUDOMONAS AERUGINOSA AZURIN  3.1  Introduction  3.1.1 Implications of Cysteinyl Radicals in Biology Cysteinyl radicals are involved in a wide assortment of biological processes. They are implicated in enzyme catalysis such as ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides to deoxyribonucleotides, providing the monomeric precursors required for DNA biosynthesis. The general proposed mechanism of RNR requires a cysteinyl radical to initiate nucleotide reduction by abstracting the 3’ hydrogen atom of the ribonucleotide substrate.224,225 Cysteinyl radicals also partake in cellular antioxidant activity. Thiols are abundant in biological systems and are considered protective and antioxidant agents. The main biological thiol is the cysteine-containing tripeptide, glutathione (GSH).  GSH acts as a  scavenger to remove free radicals formed in normal metabolism.  During the  scavenging process, a glutathiyl radical is formed by one-electron oxidation of glutathione.226,227 The importance of cysteinyl radicals is also highlighted in peptide post-translational modifications. As mentioned in chapter 1, cysteinyl radicals are precursors to several well-established cysteine modifications such as S-nitrosylation, S-glutathionylation, cysteine sulfenic acid, cysteine sulfinic acid and cysteine sulfonic  56  acid. As a result of post-translational modification such as S-nitrosylation, cysteinyl radicals are implicated in cellular signaling as well.140,228 Although cysteinyl radicals have been postulated for various important biological processes, spectroscopic evidence of protein-based cysteinyl radicals is limited.  3.1.2 Current Spectroscopic Detection The predominant method used to study radical species is electron paramagnetic resonance (EPR). EPR spectroscopy, also known as electron spin resonance (ESR), is a powerful technique for studying unpaired electrons and their interaction with their environment.  The technique is based on the same  phenomenon as NMR, the splitting of angular momentum states in the presence of a magnetic field (a.k.a., the Zeeman effect), but instead of nuclear spin states electron spin states are observed. By application of a strong magnetic field, the magnetic moment of an unpaired electron can be oriented either parallel or anti-parallel to the applied field. This creates distinct energy levels for the unpaired electrons, making it possible for net absorption of electromagnetic radiation in the form of microwaves to occur. The energy of the absorbed microwaves corresponds to the Zeeman splitting of the spin states. The field at which resonance occurs is usually reported in terms of g-values, the EPR equivalent of NMR chemical shifts.229  57  EPR has been successfully applied to detect and characterise proteinassociated radicals in numerous redox enzymes.230-232  However, protein-based  cysteinyl radicals have been shown to be difficult to observe with EPR compared to tyrosyl radicals or tryptophan radicals due to the large spin-orbit coupling of sulfur * which causes the EPR signal to broaden.233,234 Direct EPR studies of cysteinyl radicals have been carried out with low molecular-weight thiols such as cysteine and in proteins such as bovine serum albumin (BSA) and E. coli RNR (Figure 3.1).141,235,236  The EPR spectrum of the cysteine sample (Figure 3.1C) shows the  dominant feature g ⊥ at 2.008 and g at 2.29. The g component is well separated from the region of overlap with the other radicals and is a diagnostic feature of the cysteinyl radical.237  The EPR spectrum of the cysteinyl radical in BSA sample  (Figure 3.1B) shows a shift of the g component to 2.18 and a broadening. In the EPR spectrum of RNR (Figure 3.1A), the g component is broadened even more. The g component broadening causes difficulty in positively identifying a cysteinyl radical in a protein system.  *  The spin-orbit coupling constants for sulfur, oxygen, nitrogen, and carbon are 382, 151, 76, and 28 -1 233 cm , respectively.  58  Figure 3.1  First derivative X-band EPR spectra of cysteinyl radicals. A) RNR protein of E. coli (500μM); B) BSA (4 mM); and C) cysteine (300 mM). This figure has been reproduced with permission.236  3.1.3 Proposed Spectroscopic Detection Sulfur K-edge XAS is proposed as an alternative spectroscopic method for investigation and characterization of cysteinyl radicals.  Figure 3.2 shows the  possible electronic transitions that may be observed in a S K-edge XAS spectrum. Sulfide anion species having a filled valence shell will only result in direct ionization of the S 1s core electron, whereas thiolate species will have a near edge transition  59  corresponding to the SCσ* ← S 1s transition. The thiyl radical in addition to the edge jump and near edge transition will exhibit a pre-edge feature that is due to a S 3p ← 1s transition. The energy and intensity of S K-edge XAS pre-edge features will provide information on the electronic structure and reactivity of biological sulfurbased radicals.  Thus, XAS is a promising spectroscopic method in providing  information on the nature of sulfur-based radicals and their chemical behaviour. This chapter focuses on S K-edge XAS as a suitable spectroscopic method to detect cysteinyl radicals and investigates the sensitivity of S K-edge XAS to distinguish protein-based cysteinyl radicals in hydrophilic and hydrophobic protein environments.  Figure 3.2  Qualitative valence molecular orbital diagram depicting electron excitation of sulfur species: sulfide anion, thiolate and thiyl radical.  60  3.1.4 Pseudomonas aeruginosa Azurin In order to study sulfur-based radicals, isolated cysteinyl radicals were generated in Pseudomonas aeruginosa azurin (hereafter referred as azurin) using methods initially developed by Harry B. Gray and coworkers for the study of electron transfer in metalloproteins.238 Gray’s group has measured the kinetics of long-range electron transfer (~ 20 Å) reactions in metalloproteins labeled with inorganic photoinitiator complexes, coupled with laser excitation and transient spectroscopy. Azurin is involved in the respiratory chain of the bacterium P. aeruginosa where it, together with cytochrome c551, transports electrons from the membrane bound bc1 complex to a soluble nitrite reductase in the periplasmic space of the bacterium. It has been widely studied as a prototypical electron transfer protein. Azurin is a small protein comprising of a single polypeptide chain, which contains 128 amino acid residues. The redox-active metal in the wild-type protein is a redoxactive type 1 copper centre. Common to all such sites is the existence of three strongly-bound ligands: two histidines (His46 and His117) and a cysteine (Cys112), which form a roughly trigonal planar structure. In addition, azurin has two weakly interacting ligands that lie above and below the plane: the thioether sulfur of a methionine residue (Met121) and the backbone carbonyl oxygen of a glycine (Gly45).16 This metal binding site can also ligate a series of other metals, such as Co2+, Ni2+ and Zn2+.239,240 β-strands extend from the metal binding ligands forming a β sheet allowing azurin to be flexible for mutagenesis.  61  Azurin was chosen as an initial model protein because it is robust and can be easily expressed in E. coli. Also, there are different electronic environments present within the protein (Figure 3.3). This combination of properties makes azurin an attractive system in which to examine cysteinyl radicals.  Figure 3.3  Azurin model system highlighting the different electronic environments. A is hydrophilic, B is hydrophobic and C is metal binding region.  3.1.5 Cysteines of Interest in Two Distinct Regions of Azurin The structure of azurin contains two regions of interest, one hydrophilic and the other hydrophobic. In the hydrophilic region, the residue at position 108 is of interest for the cysteine radical study with an introduced surface histidine at position 107.  The surface histidine is necessary for the attachment of the photoinitiator  62  complex  as  explained  in  Section  3.3.2.  A  mutant  of  azurin  W48F/Y72F/H83Q/Q107H/Y108C (named Y108C for simplicity hereafter) was used by Gray and coworkers to study Cu1+ to Ru3+ electron transfer through the protein matrix. This mutant plasmid was kindly provided by Dr. Wehbi (past member of Gray’s group) to study the cysteinyl radical in the highly solvent accessible region. Mutations in positions 48 and 72 were made from redox-active amino acids to redoxinactive residues.  Previous studies done by Gray’s group conclude that these  mutations are necessary to prevent side electron transfer reactions from occurring. The mutation at position 83 is to ensure that no other surface histidine is available for the photoinitiator complex to attach other than to the introduced histidine at position 107. In the hydrophobic region, the residue at position 48 is of interest for the cysteinyl radical study with the available surface histidine at position 83 for the photoinitiator attachment.  A mutant plasmid W48C/Y72F/Y108F (named W48C  hereafter) was also provided by Dr. Wehbi to investigate the formation of cysteinyl radicals in the hydrophobic core. Again, mutations at positions 72 and 108 are necessary to prevent side electron transfer reactions from occurring.  Both Y108C  and W48C mutants of azurin are used to test XAS as a spectroscopic method to detect cysteinyl radicals and to probe XAS sensitivity to distinguish the cysteinyl radicals in two extreme protein environments. For quantitative analysis with S K-edge XAS, a mono cysteine protein model is necessary. Thus, mutants Y108C and W48C were further simplified by replacing all other sulfur containing amino acids such as cysteines (except the cysteine of  63  interest) and methionines. The mono Cys mutant azurin were named Y108Cm and W48Cm with single redox active cysteine in hydrophilic and hydrophobic region, respectively.  3.2  Experimental  3.2.1 Multi Site-directed Mutagenesis Using Y108C and W48C as a template for Y108Cm and W48Cm, respectively, nine additional mutations were made which are listed on Table 3.1. Polymerase chain reaction was the preferred method of choice in generating the Y108Cm and W48Cm mutant plasmids.  The Y108Cm and W48Cm mutant plasmids were  generated by Anthony Lin, (past member of Kennepohl group) prior to my joining the lab. Additional mutations in Y108Cm and W48Cm.  Table 3.1  Mutation Number Mutation Location 1  Cys3Ala  2  Cys26Ile  3  Cys112Ser  4  Met13Gln  5  Met44Gln  6  Met56Gln  7  Met64Gln  8  Met109Gln  9  Met121Gln  64  3.2.2 Sequencing To determine that the mutations were made correctly, the plasmids for the relevant mutants were over-expressed in XL1 Blue E. coli and plasmid DNA was isolated and purified using QIAprep Spin Miniprep Kit (Qiagen) with the procedures outlined in the QIAprep Miniprep handbook. Purified plasmid DNA concentration was determined using A260 and purity was confirmed using the ratio of A260/A280. Purified plasmid DNA (10 μL) with known concentration was submitted for sequencing to the Nucleic Acid Protein Service Unit (NAPS) at the University of British Columbia. This method produced a trace showing the sequence of DNA bases in the plasmid fragment of interest. By comparing the DNA sequence with the template sequence the exact composition of the resulting protein was confirmed. Y108C, W48C, Y108Cm and W48Cm were all submitted for DNA sequencing and once it was confirmed that all of these had correct sequences, protein expression was conducted. Raw output of these sequencing runs can be found in Appendix A.  3.2.3 Protein Expression and Purification Protein expression depends on transforming the appropriate plasmid into E. coli to produce protein. The commonly used cell strain for protein expression is BL21(DE3).  65  BL21(DE3) E.coli cells were thawed from -80oC to 0oC by placing in ice. Aliquots of 50 μL of thawed cells were transferred into a pre-chilled, sterile * 1.5 mL Eppendorf tube and the remainder of the cells were refrozen at -80oC.  To the  thawed cells, 1 μL of sequenced plasmid (concentration 30-60 ng) were added and gently mixed. The mixture was incubated at 0oC for 30 minutes. This mixture was then warmed to 42oC for 45 seconds followed by 5 minutes at 0oC. 250 μL of SOC medium was added to the mixture then it was placed on a shaker for 1 hour at 225 rpm at 37oC. Afterwards, 50 μL of the mixture was transferred on to a LB-agar plate containing ampicillin. The plates were incubated overnight at 37oC at which time colonies of bacteria were visible. The plate was then stored at 4oC until it was needed for the protein expression step. To express the protein on a sufficient scale, the recombinant cells were cultured on a 9 L scale. First, using a sterile toothpick two colonies were inoculated from the agar plate into 10 mL of sterile terrific broth (TB, Invitrogen) containing 60 μg/mL ampicillin. This was placed in a shaker and allowed to grow for 7 hours at 37oC, 225 rpm. After 7 hours, 1.5 mL of this starter culture was added to 6 x 4L Erlenmeyer flasks each containing 1.5 L sterile TB with 60 μg/mL of ampicillin. The remainder of the starter culture was used to prepare stock culture that was stored for later use. Starter culture (0.8 mL) and sterile 100% glycerol (0.2 mL) was placed  *  All equipment and biological media were sterilized by autoclaving. The sterilized media was cooled before adding ampicillin.  66  into a sterile microcentrifuge tube and stored at -80oC. The 4L flasks were placed in a shaker at 37oC, 200 rpm for 12 hours at which point they had reached an optical density (OD) at 600 nm of 1.2. Then, to induce protein expression, isopropyl-βthiogalacto-pyranoside (IPTG) (0.4 M, 1.5 mL) was added to each flask and incubated in the shaker at 37oC, 200 rpm for an additional 3 hours. The cells were then harvested by centrifuging the cultures at 4,000 rpm at 4oC for 20 minutes. Cell pellets were stored at -20oC until ready for protein extraction step. Osmotic shock was used as the method of choice for extraction of Y108C and W48C protein expressed in the periplasm. The cell pellets were then resuspended in a one-tenth volume of high osmolarity sucrose solution (0.3M Tris pH 8.1 with 1mM EDTA and 20% w/v sucrose). For 9 L cell cultures that were grown, 900 mL of sucrose solution was used. The suspensions were transferred to centrifuge tubes and placed on a shaker at room temperature for 30 min in order to swell the cells. The suspension was then centrifuged at 8,000 rpm at 4oC for 15 min. The swelled pellet was then resuspended in cold (4oC) 900 mL of 0.5 M MgSO4 followed by shaking at 4oC for 1 hour with the effect of lysing the cells so that the contents of periplasm can be released. The lysed cell remains were removed by centrifugation at 10,000 rpm at 4oC for 20 min. To the supernatant containing the azurin protein, 250 mM sodium acetate buffer, pH 4.5 was added to give a final concentration of 25 mM sodium acetate, pH 4.5 and this solution was left to incubate at room temperature for 20 min. A white precipitant formed at this point which was attributed to DNA.  Following the acetate precipitation, the periplasmic extrudate was  centrifuged at 8,000 rpm for 15 min. The supernatant was saved and the white  67  pellet was discarded. ZnSO4 (100 mM) was added to the supernatant to give a final concentration of 10 mM ZnSO4. This solution was left to sit overnight at 40C to allow for metal incorporation. Following the metal incorporation into azurin, the protein solution was concentrated by Amicon filtration over a 10,000 MWCO membrane (YM-10 Millipore). The concentrated protein solution was then washed several times with 25 mM sodium acetate pH 4.5 buffer until the effluent absorption at 260 nm was close to zero (small bits of DNA having washed through). The concentrated azurin was collected and stored in the acetate buffer at 4oC until the purification step. The preferred method for purification of crude azurin sample is purification with cationic exchange resin (HiTrapTMSP HP, GE Healthcare).  The resin consists  of a surface sulfonate, thus it has an affinity for positively charged species. Given the pI of azurin is 5.4 and the protein solution was in acidic media (pH 4.5), the azurin would have a positive net charge which bound tightly to the SP column. The bound protein was eluted with a gradient between buffer A (25 mM sodium acetate buffer, pH 4.5) and buffer B (300 mM sodium acetate buffer, pH 4.5). First, the protein solution was applied to the SP column in 5 mg batches. Once the protein was loaded to the column, the loaded column was washed with 20 mL of buffer A to remove any unbound protein. This was then followed by a single elution step of the azurin protein with 85% buffer A, 15% buffer B. After the azurin had been eluted, the remaining protein impurities were eluted from the column with 100% buffer B.  68  To confirm the purity of the eluted azurin, the collected azurin fraction was analysed with SDS-PAGE. The eluted azurin sample (~ 50 μM, 5 μL) was mixed with 5 μL of denaturing buffer (consisting of 0.5 M Tris, pH 6.8, 10% w/v SDS, 10% v/v β-mercaptoethanol, 0.5% w/v bromophenol blue, and 20% v/v glycerol). This mixture was loaded on to the gel along with 10 μL of molecular weight marker in a separate lane. The gel was then developed by standard techniques and stained to show bands of approximate molecular weight.  3.2.4 Synthesis of Rhenium Complex In order to generate controlled cysteinyl radicals in azurin, a photoinitiator complex (Figure 3.4) was synthesized and attached to the azurin. Synthesis of the photoinitiator  complex  was  based  on  previously  published  experimental  procedures.241,242 First, 0.4 g of 5,6-dimethyl-1,10-phenanthroline (dmphen, from Aldrich) was dissolved in 15 mL of toluene in a 25 mL round bottom flask equipped with stir bar.  Then, 0.5 g of rhenium pentacarbonyl chloride (Re(CO)5Cl, from  Aldrich) was added to the flask and this mixture was heated to 60oC. In order to exclude water vapour, the flask was equipped with a reflux condenser. The mixture was allowed to stir for 5 hours at 60oC. During this time, the solution changed colour from white to dark yellow with the formation of a yellow precipitate. The yellow precipitate is Re(CO)3(dmphen)Cl which is insoluble in toluene. The suspension was then filtered over a fine porosity glass fritted funnel to give a yellow solid which was then washed several times with toluene. To remove the halide in the complex, the solid was then dissolved in 30 mL of dichloromethane.  To this solution,  69  trifluorosulfonic acid (also known as triflic acid, from Fluka) was added dropwise until the mixture became transparent.  Triflic acid (HOTf) is used to protonate the  coordinated chloride and promote dissociation from the Re centre. Hydrochloric acid bubbled off and the triflate substituted for Cl. The reaction was left to stir at room temperature for 4 hours. Then diethylether was added dropwise to this solution causing a yellow precipitate to form which was Re(CO)3(dmphen)OTf. This yellow solid was collected by filtering through fine porosity filter and washed with ether. This solid was then added to 25 mL water in a beaker equipped with stir bar. Water underwent a ligand substitution reaction to displace the triflate. The solution was stirred for 2 hours at 60oC then the solution was filtered over a medium porosity glass fritted funnel. The solution contained the Re(CO)3(dmphen)(H2O)+. The solid was collected and the triflate displacement was repeated twice more.  All the  solutions were then collected and the water was removed by gentle boiling followed by cooling the mixture to room temperature. The [Re(CO)3(dmphen)(H2O)+](OTf-) precipitated as orange crystals. These crystals were stored until needed for rhenium labelling reactions.  To confirm the identity of the crystals, the crystals were  submitted for MALDI-TOF analysis at the Mass Spectrometry lab in UBC. The result indicated a m/z peak at 479.3 confirming Re(CO)3(dmphen) structure. O N C C O Re C O N OH2  Figure 3.4  Re-photoinitiator complex  70  3.2.5 Re-labeling Reaction Before labeling, the protein was equilibrated in 25 mM HEPES buffer, pH 7.2 (Sigma-Aldrich). The protein solution was then concentrated using Amicon Ultra-15 centrifugal filter with 10 kDa MWCO (Millipore) to ~3.5 mM. Meanwhile, 0.0084 g of [Re(CO)3(dmphen)(H2O)+](OTf-) was dissolved in 6.5 mL of water by gentle heating and stirring over 2 hours to give a ~2 mM solution. The concentrated protein sample was divided in 5 aliquots of 100 μL each into a 2 mL Eppendorf tubes. To this, 1.3 mL of Re-complex solution was added. These tubes were incubated at 37oC in the dark for 7 days. At the end of this time, the labeled samples were concentrated with Amicon centrifugal filter. The Re-labeled azurin was then exchanged into 25 mM sodium acetate buffer, pH 4.5 using the PD-10 gel filteration column (Pharmacia). The collected protein was again concentrated by Amicon centrifugal filter and allowed to stand in the dark at 4oC for 3 days. Acetate buffer allowed the rhenium that might be mislabeled at glutamates or aspartates residues to be pulled off. Then, the Re-labeled azurin was centrifuged to remove the rhenium precipitate that did not bind and was prepared for the protein purification step. Using PD-10 column equilibrated the protein in 20 mM sodium phosphate, 750 mM sodium chloride, pH 7.2.  3.2.6 Purification of Labeled Azurins Re-labeled azurin purification was necessary to remove proteins that are not labeled at the single surface histidine. To accomplish this, a HiTrapTM Chelating HP column (GE Healthcare) was employed. The column uses a surface composed of  71  histidines which can be charged with Cu2+ from a CuSO4 solution that was injected prior to loading the protein.  The resin bound Cu2+ can bind tightly to a single  exposed histidine residue. Thus if the azurin was not labeled with the rhenium then the surface histidine would be available to bind to the resin bound Cu2+ and the labeled protein will pass through the column. The protein was applied to the column in 5 mg batches and eluted with 20 mM sodium phosphate, 750 mM sodium chloride, pH 7.2. To remove the tightly bound unlabeled protein, the column was washed with a solution containing 20 mM sodium phosphate and 750 mM ammonium chloride, pH 7.2.  The unlabeled protein fraction was recovered and  reused for labelling reactions. Both Re-Y108C and Re-W48C were submitted for MALDI-TOF analysis to ensure that proteins were labeled.  3.2.7 The Flash/Quench/Freeze Methodology The following procedure was used to generate the cysteinyl radicals in ReW48C and Re-Y108C. The purified Re-labeled protein was reduced in volume to 150 μL to give final concentration of ~7-8 mM. The concentrated protein solution was incubated with a small amount of oxidative quencher, [Co(NH3)5Cl]Cl2 for 5 min at room temperature in the dark. The solution was then carefully removed from the remaining solid by pipette and transferred to a microcentrifuge tube. To this 100 μL of 100% glycerol was added to give final concentration of 40% glycerol (v/v) in the sample.  Glycerol is a good glassing agent which is a prerequisite for solution  sample in soft XAS data collection. The protein solution was then degassed using a Schlenk line and purged with Ar gas. This pump/purge procedure was repeated  72  several times to ensure complete removal of oxygen.  In an Ar-filled inert  atmosphere glovebag, the sample was then transferred to an XAS cell sealed with a polypropylene window on the front. The XAS cell containing the protein sample was exposed to a Xe arc lamp for 10 seconds and then frozen in liquid nitrogen. This allows for trapping of photogenerated cysteinyl radicals.  3.2.8 XAS Data Acquisition S K-edge XAS data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 6-2 under ring conditions of 3 GeV and 60-100 mA. The setup used a 54-pole wiggler beamline operating in high-field (10 kG) mode with a Ni-coated harmonic rejection mirror and a fully tuned Si(111) double-crystal monochromator. Energy calibrations were carried out using an external standard of sodium thiosulfate (Na2S2O3) with the first pre-edge feature being calibrated at 2472.02 eV. Signals were detected with a N2 fluorescence (Lytle) detector at the temperature of liquid He.  For each sample two sweeps were taken and the  experiment was repeated twice to ensure reproducibility.  3.2.9 XAS Data Processing and Analysis SixPACK software was used for XAS data processing.220 Pre and post-data calibration scans were compared, and the energy scale of the data was adjusted by taking the first pre-edge feature of Na2S2O3 and setting this to 2472.02 eV. Background subtraction and normalization were performed simultaneously using a linear pre-edge and post-edge function.  73  The commercially available PeakFit software package243 was used to fit the preedge and near-edge feature with single pseudo-Voigt line shape (Equation 3.1). This line shape is appropriate as the experimental features are expected to be a Lorentzian transition and a Gaussian line shape is imposed by the beam line optics. A fixed 1:1 ratio of Lorentzian:Gaussian shapes were used for pre-edge and nearedge features.  The ionization edge feature was fitted with a cumulative  Gaussian/Lorentzian function (Equation 3.2).  The spectra were fitted over the  energy region from 2467 eV to 2476 eV. Equation 3.1 Voigt Area Function ∞  a0  y=  ∫  exp( −t 2 )   x − a1  −∞ 2 a3 +  −t  a2  ∞ 2 exp( −t ) ∫−∞ a32 + t 2 dt  2  dt  a0 = area a1 = centre a2 = width (>0) a3 = shape (≥0)  Equation 3.2 Cumulative Gaussian/Lorentzian Function  y = a4 ×   x − a  a0  a 1 1 + erf    + (1 − a4 ) × 0  a  π 2  2      −1  x − a1  π   tan  +   a3  2    a0 = amplitude a1 = inflection point a2 = Gaussian width a3 = Lorentzian width a4 = shape  74  3.3  Results and Discussion  3.3.1 Re-labeled Azurin mutants Mutants Y108C and W48C were successfully expressed in E. coli and purified. The purity of the mutant azurin proteins were confirmed by the SDS gels that showed only a single band of approximate weight of 14 kDa (Figure 3.5).  Figure 3.5  SDS-PAGE Gel. Lane 1 is protein ladder, lanes 2-7 are purified fractions of Y108C, and lane 8 is Y108C isolated by osmotic shock.  Y108Cm and W48Cm however, were not successfully expressed.  Several  attempts were made to express these mutants under different conditions such as various temperatures, ODs at which the expression was induced, and the IPTG concentrations.  Table 3.2 lists the conditions that were tried. Of these conditions,  protein expression of Y108Cm and W48Cm at 28oC and 0.8 OD with 0.8 mM IPTG concentration showed the most promising results, but the expression level was not  75  enough to proceed further. Therefore, only Y108C and W48C were expressed and purified for incorporating Re-complex.  Table 3.2  Protein expression conditions attempted for Y108Cm and W48Cm. highlighted condition produced observable protein expression level.  Temperature (oC)  Optical Density  IPTG Concentration (mM)  22  0.8  0.4  22  0.8  0.8  22  1.0  0.4  22  1.0  0.8  22  1.2  0.4  22  1.2  0.8  28  0.8  0.4  28  0.8  0.8  28  1.0  0.4  28  1.0  0.8  28  1.2  0.4  28  1.2  0.8  33  0.8  0.4  33  0.8  0.8  33  1.0  0.4  33  1.0  0.8  33  1.2  0.4  33  1.2  0.8  37  0.8  0.4  37  0.8  0.8  37  1.0  0.4  37  1.0  0.8  37  1.2  0.4  37  1.2  0.8  The  76  Histidine acts as an ideal ligand for coordination compounds. Thus, a single surface histidine on each azurin mutant binds to a Re-complex. Surface histidine at position 83 is exploited for the attachment of the Re-complex in W48C, and for Y108C the introduced surface histidine at position 107 is used.  The histidine  attaches to the Re(CO)3(dmphen) unit by displacing water from the labeling reagent, [Re(CO)3(dmphen)(H2O)+](OTf-). In order to determine if a single Re-complex is attached to the mutant azurin, the Re-labeled azurin was submitted for MALDI-TOF (spectrum found in Appendix B). The results are shown in Table 3.3 along with an unlabeled sample.  The difference in the labeled and unlabeled protein is Δm/z  479 which is the molecular weight of Re(CO)3(dmphen). This indicates that a single rhenium is attached in both Y108C and W48C.  Table 3.3  Mass spectral results for the labeled and unlabeled azurin mutants.  Azurin mutant  Unlabeled  Labeled  W48C  13 828.8  14 308.4  Y108C  13 824.7  14 304.5  3.3.2 Generation of Cysteinyl Radicals One of the most efficient methods of quickly inducing a change in redox states is the so-called flash/quench technique.244  This method is based on  irradiation of a molecule, in this case a Re-complex, to generate a reactive excited state. The excited state can then interact with an exogenous electron acceptor [Co(NH3)5Cl]2+ to generate a highly reactive redox Re(II). The exogenous electron  77  acceptor is referred to as an oxidative quencher. After the flash/quench, the photooxidized Re-complex will react with an electron donor in the system. Since the Recomplex is conveniently attached near a Cys residue (~10 Å), Cys can donate an electron to Re(II) and form a cysteinyl radical. A representation of this experiment is shown in Figure 3.6.  Figure 3.6  Simplified flash/quench reaction between Re-complex and the nearby Cys with oxidative quencher (Qo).  3.3.3 Characterization of Cysteinyl Radicals by Sulfur K-edge XAS A small but noticeable pre-edge feature was present in the S K-edge XAS spectra of the W48C radical and Y108C radical. The pre-edge feature arises from transitions to empty or partially empty valence orbitals. The XAS spectra of W48C and W48C radical (Figure 3.7) showed an intense near-edge transition at 2473.6 eV that corresponds to the SCσ* ← S 1s transition and the W48C radical showed a pre-  78  edge feature at 2469.3 eV which corresponds to the S 3p ← 1s transition due to the presence of a hole in the S 3p orbital. The presence of such a pre-edge feature is characteristic of species that exhibit a radical character on the sulfur atom. Similarly, the XAS spectrum of Y108C radical (Figure 3.8) showed a pre-edge feature at 2470.9 eV in addition to the near-edge feature. The 1.6 eV shift in energy of the preedge feature for Y108C radical is due to the cysteinyl radical being in a hydrophilic environment that is susceptible to interact with the H2O. This interaction with H2O results in hydrogen bonding (H-bonding) which may play an important role in tuning the electronic structure and reactivity of the cysteinyl radical. H-bonding is likely to increase the energy of the 3p orbital in which an unpaired electron resides hence increasing the pre-edge transition energy. Also, the dielectric constant of the local protein environment of the Cys radical in Y108C can cause the observed energy change.  The cysteine in W48C, on the other hand, is buried in the protein  environment, thus it is well protected from H2O and therefore H-bonding is not in effect. The effect of H-bonding is also observed in the pre-edge features that result from metal ligated thiolate.245 Solomon and coworkers have studied a series of P450 model complexes with increased H-bonding using S K-edge XAS. In addition to the increase in energy of the pre-edge transition, a decrease in intensity is observed in complexes of H-bonding. This decrease in intensity of the pre-edge feature is also evident in Y108C radical compared to the W48C radical.  The  intensity of the pre-edge feature (peak area) is quantitatively estimated from fits to the experimental spectra (Figure 3.9). The intensity of the pre-edge feature for the  79  Y108C radical and W48C are 0.23 and 0.31, respectively. However, the pre-edge intensity of the radical is dependent on the radical yield which cannot be quantified in this model protein system. In order to estimate the radical yield, a mono Cys protein system is required. Thus, the intensity is coincidently lower for the cysteinyl radical in the hydrophilic region. However, the observed energy difference of the pre-edge feature of Y108C radical compared to the W48C radical suggests that the combined effects of hydrogen bonding and the dielectric constant of the local protein environment are likely to play a role.  Figure 3.7  S K-edge XAS spectra of W48C and W48C radical with pre-edge feature expanded (inset).  80  Figure 3.8  S K-edge XAS spectra of Y108C and Y108C radical with pre-edge feature expanded (inset).  Figure 3.9  S K-edge XAS experimental data and fit for W48C radical.  81  3.4  Conclusions Although protein-based cysteinyl radicals are implicated in various biological  functions, direct detection has been challenging.  EPR is the predominant  spectroscopic method in studying unpaired electron, but it is difficult to detect cysteinyl radicals due to the large spin-orbit coupling of sulfur that results in signal broadening. My work in generating controlled cysteinyl radical in azurin has been successful in investigating with sulfur K-edge XAS. The transition of the 1s electron into the partially filled 3p orbital results in a pre-edge feature, that is well separated from the near-edge feature. Thus, S K-edge XAS has been shown to be a promising spectroscopic method in investigating sulfur-based radicals. In addition, S K-edge XAS is sensitive in differentiating the cysteinyl radical in different protein environments. A cysteinyl radical in a hydrophilic environment is able to H-bond, which can affect the reactivity of this radical and is evident in the pre-edge feature energy.  82  CHAPTER 4: PHOTOCHEMISTRY OF METHIONINE, METHIONINE SULFOXIDE, AND METHIONINE SULFONE  4.1  Introduction  4.1.1 Methionine Oxidation Formal oxidation of methionine is generally considered to involve two oxo transfer steps, producing methionine sulfoxide (MetSO) in the first oxidation step and methionine sulfone (MetSO2) in the subsequent step (Scheme 4.1). While the first oxidation product MetSO can be physiologically reduced back to Met by methionine sulfoxide reductase (Msr), a further oxidation to MetSO2 is considered to be biologically irreversible, as highlighted in chapter 1.246 Oxidative damage resulting in Met oxidation in proteins has been linked to several diseases such as age-related cataracts and Alzheimer’s disease.174,247  R  O  H N  R'  S  Met  Scheme 4.1  Oxidation  R  H N  O R'  Oxidation  R  Reduction S  O  MetSO  H N  O R' S O O  MetSO2  Oxidation and reduction of methionine residues in proteins.  83  4.1.2 Age-related Cataracts Cataracts are an eye disease that typically progresses slowly to cause vision loss and is potentially blinding if untreated; it is a leading cause of blindness in the world.202 Age-related, or senile cataracts, are the most common type of cataracts. The term “cataract” has been used to mean any opacity or loss of transparency of the lens. Lens opacities are the earliest visible changes in cataractogenesis. The lens, the biconvex crystalline structure behind the iris, serves to transmit, filter and focus light upon the retina. To serve this function, the eye lens has to provide transparency and a high refractive index.  The transparency and high refractive  index of the lens are due to the high concentration of structural proteins called crystallins.248  Crystallins are known to constitute about 90% of water soluble  proteins of the lens and can be separated into three distinct families, the α-, β-, and γ-crystallin, on the basis of their oligomeric size. In addition to its structural role, αcrystallins has been shown to function as a molecular chaperone, helping to prevent formation of large light-scattering aggregates from denatured crystallins.205 To maintain transparency, the crystallins must remain stable for the duration of the host’s lifetime without being denatured.  However, with age, crystallins  undergo a wide variety of irreversible covalent modifications, caused by proteolysis, deamidation, extensive oxidation and cross-linking.249  Such undesired post-  translational modification of the crystallins is believed to decrease their solubility in the lens, which leads to cataract formation.  Oxidation of α-crystallin has been  demonstrated to cause a significant reduction in its chaperone activity, which suggests that oxidation may have important consequences for protein aggregation in 84  the lens.206  One of the major post-translational modifications of α-crystallin is  oxidation of Met to MetSO and MetSO2. Met oxidation in both bovine and human αcrystallin occurs in vivo.247,249  Several factors have been attributed to cataract  formation. Of these, UV light exposure has been proposed to be involved in agerelated cataract formation.250  In general, very little UV-C (< 280 nm) radiation  reaches the earth’s surface because of atmospheric absorption in the ozone layer. However, UV-B (280-320 nm) and UV-A (320-400 nm) can reach the eye, and the human cornea filters out all radiation below 290 nm. UV-B radiation is generally absorbed by the cornea and aqueous humor before reaching the lens,251 yet UV-B exposure over extended periods has been shown to cause damage to the lens that can lead to cataract formation.252-254 The higher wavelength UV-A can pass through the cornea to be absorbed by the lens, thus it has also been proposed to cause damage to the lens leading to cataract formation. 255,256 However, the mechanism of light-induced cataract formation is still not clear, in particular the light-induced oxidation of Met residues.  4.1.3 Photooxidation of Thioether Efforts have been made in studying the mechanism of sensitized photooxidation of functionalized thioethers.257,258  Under such conditions, the  mechanism of photooxidation is believed to involve the formation of singlet oxygen (1O2), as shown in Scheme 4.2. 1O2 produced on energy transfer from the sensitizer reacts efficiently with dialkyl sulfides to yield persulfoxide intermediates.  The  persulfoxide acts as an oxygen atom transfer agent to form the sulfoxide259,260 and  85  sulfone261 or can undergo fragmentation, however, the mechanisms of these formations are not clear.  An alternative mechanism involves photosensitized  electron transfer (ET) oxidation (Scheme 4.3), where a sulfide radical cation forms upon ET to the excited sensitizer, which is then regenerated by a reaction with oxygen to yield the superoxide anion.261 Recombination of the sulfide radical cation and the superoxide anion yields the persulfoxide intermediate which then can react with another thioether to form sulfoxide. O R S R O  + R2SO  R2SO  R2S +  1  O O R S R  O2  Fragmentation Products  R2S  O 2 R S R  Scheme 4.2  Proposed mechanism of photooxidation of thioether involves the formation of singlet oxygen.259  1  Sens* + R2S Sens  O2 Scheme 4.3  + O2  + R2S  Sens O2  + R2S + Sens  O O R S R  R2SO  Alternative mechanism of photooxidation of thioether involves formation of sulfide radical cation.261  86  The photochemistry of methionine and its related species methionine sulfoxide and sulfone have not been explored in the absence of sensitizer. Thus, this chapter provides new insights to factors that affect the photochemically activated Met and MetSO redox process in the presence and absence of oxygen.  4.2  Experimental  4.2.1 Materials L-Methionine (purity 98.0%) and L-methionine sulfoxide (purity 99.0%) were purchased from Sigma-Aldrich. L-Methionine sulfone (99.0%) was purchased from MP Biomedicals. These compounds were checked for contamination using ESI and NMR before use.  Sulfur-free Kapton tape was purchased from Creative Global  Services and checked for sulfur contamination using sulfur K-edge XAS before use.  4.2.2 Sample Preparation Solid samples of Met, MetSO, and MetSO2 were finely ground, placed on Kapton tape, and mounted across the window of an Al plate. Solution samples of Met, MetSO and MetSO2 were prepared at a concentration of 10 mM in distilled water with 50% glycerol as a glassing agent to reduce diffraction produced by ice crystals in XAS. For aerobic photochemical experiments, samples were exposed to irradiation from a 75 W Xe arc lamp for 0-3 hours at room temperature followed by analysis using XAS. Anaerobic photochemical experiments were performed in a He filled glovebag, and XAS data were collected in situ (Figure 4.1).262 An open-flow liquid He cooler from Cryo Industries allowed data to be obtained at ~ 20 K. For 1H  87  NMR analysis, ground samples were irradiated on a clean Petri dish and then dissolved in D2O for spectroscopic analysis.  1  H NMR spectra were collected on a  Bruker Avance 400 MHz spectrometer at ambient temperature.  All of the  photochemistry was performed in the solid state or in 10 mM aqueous solutions, with qualitatively identical results.  Figure 4.1  Sample compartment for in situ anaerobic photochemistry setup at SSRL beamline 6-2, where I0 is the incident X-ray beam.  4.2.3 XAS Data Acquisition XAS data were collected at the SSRL on beamline 6-2 under conditions described in section 3.2.8.  4.2.4 Data Processing and Analysis SixPACK software was used for XAS data processing.220 Pre- and post-data calibration scans were compared, and the energy scale of the data was adjusted by  88  taking the first pre-edge feature of Na2S2O3 and setting this to 2472.02 eV. Background subtraction and normalization were performed simultaneously using a linear pre-edge and post-edge function. SixPACK software was also used for principal component analysis (PCA), target transformation, and linear least-squares fitting. PCA was used to determine the smallest number of principal components (PCs) required to sufficiently describe the composite data set.  During target transformation, SPOIL value ranges as  defined by Malinowski263 were interpreted as follows: acceptable (<3), moderately acceptable (3-6), and unacceptable (>6). Linear least-squares fitting of reference spectra was used to evaluate component spectra over the energy range from 2465 to 2490 eV.  4.3  Results and Discussion  4.3.1 Met Photochemistry Photoirradiation of Met by Xe arc lamp under aerobic conditions produces rapid photodegradation of the thioether. The changes can be observed in the S Kedge XAS spectra as a function of irradiation time, (Figure 4.2). The intense main feature of Met at ~2473.5 eV corresponds to the thioether CS σ*← S 1s transition.264 Upon irradiation, this feature decays with formation of new, intense features at 2476.3 eV and 2479.9 eV.  These new features correspond to the formation of  MetSO and MetSO2 as seen in Figure 4.3 of S K-edge XAS spectra of clearly resolved features for MetSO (2476.3 eV), and MetSO2 (2479.9 eV).  At longer  89  irradiation times, further changes are observed, including the appearance of higherenergy features at 2481.0 eV and 2482.5 eV. These more highly oxidized species were presumed to result from fragmentation via carbon-sulfur bond cleavage. The feature at 2481.0 eV was consistent with the formation of sulfenate species, given its similarity to the pre-edge feature of methane sulfenate (CH3SO3-) in Figure 4.3. Similarly, the feature at 2482.5 eV corresponds to the highly oxidized form of sulfur; sulfate (SO42-).  Liebster and co-workers in their work on radiation induced Met  degradation have identified the fragmentation products of Met to be homocysteic acid and homoserine.265 sulfate species.  The formation of homoserine is proposed to result in  Thus it is likely that the feature at 2481.0 eV is due to the  fragmentation of Met resulting in homocysteic acid and the feature at 2482.5 eV to be a sulfate.  Figure 4.2  S K-edge XAS spectra with photoirradiation of Met by Xe arc lamp under aerobic conditions.  90  Figure 4.3  S K-edge XAS spectra of Met, MetSO, MetSO2, CH3SO3-, and SO4-2.  Under anaerobic conditions, Met photoirradiation by Xe arc lamp does not produce MetSO or MetSO2 (Figure 4.4), presumably because of the lack of an oxygen source. However, careful investigation of the time-resolved XAS spectra indicates the formation of a small pre-edge feature at low energy ~ 2470.0 eV (Figure 4.4 inset). The formation of this pre-edge feature is concomitant with a small decrease in the CS σ*← S 1s feature of Met, which eventually achieves an apparent photo-steady state (Figure 4.5). My work with azurin cysteinyl radicals (see Chapter 3) and other related work266 suggests that such low-energy pre-edge features are consistent with the formation of sulfur-centred radical species.  Therefore, this  feature is tentatively assigned to the formation of the one-electron-photooxidized  91  •  species MetS +. Evidence for the Met radical cation has been previously observed in pulse radiolysis reaction of N-acetyl-L-methionine amide.167 Formation of a photosteady state during XAS data collection can easily result from concomitant X-ray photoreduction of the cation radical to generate Met, as shown in Scheme 4.4.  Figure 4.4  S K-edge XAS spectra with in situ photoirradiation of Met by Xe arc lamp under anaerobic condition. Inset shows the difference spectra of the lowenergy.  92  Figure 4.5  Kinetic profile of the weak pre-edge feature from in situ photoirradiation of Met by Xe arc lamp under anaerobic conditions.  Xe arc Met  MetS X-ray  Scheme 4.4  Met photooxidation by Xe arc lamp and photoreduction by X-ray beam.  The mechanism of aerobic photooxidation of Met is generally believed to involve photoexcitation of dioxygen to its highly energetic singlet excited state followed by reaction with the thioether. The presence of sensitizer may enhance the production of 1O2 as well as provide alternative or concurrent pathways in which the Met radical cation is generated (Scheme 4.3).  My work on the anaerobic Met  photochemistry suggests that a third option must be considered in which direct one-  93  electron photoexcitation of Met occurs in the absence of an exogenous sensitizer. Given the small absorption coefficient for Met in the visible region (Figure 4.6), this is rather unexpected. However, high-resolution electronic spectroscopy reveals weak low-lying absorption bands at 262 and 285 nm assigned as singlet to triplet transitions.267,268 I postulate that these triplet excited states are sufficiently high in energy (~4 eV) to make them efficient reductants, thus yielding the cation radical. Reactivity through this triplet-excited-state channel may also point to an additional pathway for O2 photoactivity. The formation of a 3Met* species should allow for a rapid spin-allowed reaction with ground-state 3O2. The two new pathways that are proposed here have not been explored and provide an alternative mechanism for Met oxidation.  Figure 4.6  UV-Vis spectra of Met, MetSO, and MetSO2.  94  4.3.2 MetSO Photochemistry The MetSO photoirradiation by Xe arc lamp under aerobic conditions is shown in Figure 4.7. As with Met photoirradiation, formation of MetSO2 is observed at relatively short times. At longer irradiation times, more highly oxidized species of sulfenate and sulfate that are likely due to fragmentation products are observed. However, MetSO photoirradiation also results in the formation of a new feature at lower energy, indicative of photoreductive processes. An intense feature at 2473.5 eV, corresponding to the spectrum of the parent thioether Met is observed during photoirradiation.  Figure 4.7  S K-edge XAS spectra with photoirradiation of MetSO by Xe arc lamp under aerobic conditions.  95  The MetSO photoirradiation by Xe arc lamp in the absence of O2 shows no evidence of oxidation products (Figure 4.8). However, the lower-energy feature at 2473.5 eV is still observed with a simultaneous decrease in the intensity of the MetSO feature. Photoreduction is also observed as a result of photodamage from the X-ray beam during data collection, but the rate of formation is markedly smaller than the observed irradiation from the Xe arc lamp, (Figure 4.9). To confirm that the feature at 2473.5 eV is identical to that of Met, 1H NMR analysis was performed on the photoirradiated MetSO by Xe arc lamp (Figure 4.10). The signal from the methyl group protons (S-CH3,δ = 2.04) is useful in identifying the formation of Met in the photoirradiated MetSO.  Figure 4.8  S K-edge XAS spectra with in situ photoirradiation of MetSO with Xe arc lamp under anaerobic conditions.  96  Figure 4.9  S K-edge XAS spectra with X-ray photoreduction of MetSO during data collection.  A more quantitative analysis of the photoreductive process was obtained through principal component analysis (PCA) and target transformation to identify the number and types of components, respectively. From the PCA, it was identified that the first two PCA components of the XAS spectra of irradiated MetSO accounted for over 99% of the total variance (Table 4.1). The first two components reproduce all of the features of the spectra, confirming that anaerobic irradiation of MetSO yields only one product. Based on the PCA results, target transformation was performed using two components in order to identify the two species.  According to the  calculated SPOIL values (Table 4.2) and by visual inspection of the target transforms, the species were identified as MetSO and Met. Linear least-squares  97  fitting of Met and MetSO in fractional contributions of 0.46 and 0.54, respectively yielded a reasonable fit to the MetSO irradiated data under anaerobic condition (Figure 4.11).  Figure 4.10  1  H NMR spectra of MetSO photoirradiation with Xe arc lamp products under anaerobic conditions after 15 minutes.  98  Table 4.1  Results from principle component analysis of in situ photoirradiation of MetSO by Xe arc lamp under anaerobic conditions. The highlighted portion shows the minimum number of components needed for >99% cumulative variance.  Component Eigenvalue  Cumulative Variance  1  52.378  0.924  2  3.975  0.994  3  0.159  0.997  4  0.050  0.998  5  0.025  0.998  6  0.014  0.999  7  0.012  0.999  8  0.010  0.999  9  0.009  0.999  10  0.008  1.0  Table 4.2  Results of PCA target transformation performed in SixPACK  Target (Standard)  SPOIL  Cysteine  7.12  Methionine  3.09  Methionine Sulfoxide  2.26  Methionine Sulfone  14.57  99  Figure 4.11  Linear least-squares fit of in situ MetSO photoirradiation by Xe arc lamp under anaerobic conditions after 90 minutes.  Harvey and Lang269 have reported a similar photoreduction of DMSO to DMS. They attributed disproportionation as a likely source of DMS; however, the observation of MetSO irradiation by Xe arc lamp under anaerobic conditions is inconsistent with this interpretation as no MetSO2 was observed, whereas Met was still produced. To ensure that the photochemistry of DMSO is similar to MetSO, DMSO was irradiated by Xe arc lamp under anaerobic conditions and analysed by XAS, (Figure 4.12). The formation of the photoreduced species, DMS was observed with concomitant decrease in DMSO feature showing that DMSO photoreduction is indeed proceeding in a similar manner to MetSO.  100  Figure 4.12  S K-edge XAS spectra with in situ photoirradiation of DMSO by Xe arc lamp under anaerobic conditions.  4.3.3 MetSO2 Photochemistry Photoirradiation of MetSO2 by Xe arc lamp is both oxidatively and reductively inert under aerobic and anaerobic conditions (Figure 4.13). A small background reaction was observed and the products were identified as resulting from MetSO contamination undergoing photoreduction. The lack of highly oxidized species such as sulfenate and sulfate due to fragmentation suggests that MetSO2 is highly stable. Also, the fragmentation products such as sulfenate and sulfate that are observed in  101  Met and MetSO photoirradiation are likely to go through the persulfoxide intermediate postulated by Foote and co-workers.259  Figure 4.13  4.4  S K-edge XAS spectra with photoirradiation of MetSO2 by Xe arc lamp under aerobic conditions.  Conclusions Photochemical redox processes of Met are particularly relevant in cataracts,  whose formation is correlated with long-term UV irradiation of the crystalline lens.250 Physiologically irreversible oxidation of Met to MetSO2 should lead in many cases to the loss of function and, therefore, to pathophysiological situations. Evidence of  102  MetSO2 has been reported in age-related cataracts. Also, Met oxidation to MetSO in combination with insufficient reduction capacity to Met has similar consequences. There is significant literature on the photoredox properties of Met in the presence of sensitizers in order to enhance 1O2 production. My work shows the photochemistry of Met in the absence of such sensitizers which give rise to two new pathways. First, a direct one-electron oxidation of Met to give a sulfur-centered radical cation that can react with oxygen to give the oxidized species, MetSO and MetSO2. Second, formation of 3Met* can go through a spin allowed reaction with molecular oxygen to produce MetSO and MetSO2. The human eye is potentially vulnerable to solar radiation derived photodamage, although there are several mechanisms that protect the eye from such damage.270 Since the maximum absorption of the lowestenergy singlet to triplet transition in Met (285 nm) extends into the 300-400 nm range, it may be possible that, over long time frames, this overlap may cause photodamage. The photoreduction of MetSO back to Met also suggests that in the absence of O2, oxidative damage involving MetSO may potentially be repaired. MetSO2 is a photochemical dead-end product, thus the formation of sulfone may ultimately be the most detrimental product of photooxidation.  103  CHAPTER 5: METAL-INDUCED METHIONINE OXIDATION IN AMYLOID-BETA PEPTIDE  5.1  Introduction  5.1.1 Alzheimer’s Disease Alzheimer’s disease (AD) is a neurodegenerative disease characterized by a progressive decline in memory, impairment of cognitive function and behavioural deterioration.271 Currently, there are two types of AD recognized: early-onset and late-onset AD. Early-onset AD is diagnosed before the age of 65 and genetic factors have been identified in its development.272 Late-onset AD is the most common type of the disease, affecting about 95% of all those with AD. It affects people over the age of 65 and advancing age is the major risk factor.273 The pathological hallmarks in both forms of AD are extracellular amyloid plaques and intracellular neurofibrillary tangles.168-170 The main component in the plaques is a peptide called amyloid-β (Aβ) and the tangles are made of hyperphosphorylated tau protein.  How these  features interrelate and which one is of primary importance is still unclear. However, one of the most prevalent hypotheses is the amyloid cascade hypothesis postulated by Hardy and Higgins in 1992.274 It proposes that the fibrillisation of amyloid-β peptide as amyloid deposits is critically responsible for the neurodegeneration that occurs in AD. This prominent hypothesis has guided most of the AD research and provided the framework for the development of therapies used to treat AD.  104  5.1.2 Amyloid-beta (Aβ) Peptide Aβ peptide is found ubiquitously in human cells, but its natural function is not yet well understood. It is generated by sequential proteolytic cleavage of the much larger amyloid precursor protein (APP), a transmembrane protein of unknown function with single membrane-spanning domain. APP can be processed by α, β, and γ-secretases; Aβ is generated by successive action of the β and γ-secretases, (Figure 5.1). β-Secretase cleaves to form the N-terminal end of the Aβ, while γsecretase produces the C-terminal end.  γ-Secretase cleaves within the  transmembrane region of APP and the cleavage site determines the length of Aβ peptide. γ-Secretase cleaves to give Aβ1-40 and Aβ1-42 which are 40 and 42 amino acids in length, respectively. The two additional amino acids in Aβ1-42 are isoleucine and alanine (Figure 5.2) which are hydrophobic amino acids, which therefore has a higher propensity for aggregation. Aβ peptides consist of a largely hydrophilic Nterminal domain (residues 1-28) and a hydrophobic C-terminal domain (residues 2940/42).  Figure 5.1  Processing of the amyloid precursor protein (APP) to yield amyloid-β (Aβ) peptide.  105  Figure 5.2  Amino acid sequence of Aβ1-42. Note that the peptide contains a single methionine residue at position 35.  Aβ has been found in three biochemical forms in the brain:  membrane  associated, soluble and aggregated. In healthy individuals, the vast majority of Aβ is membrane associated, but in individuals with AD the aggregated and soluble fractions increase markedly.275,276 The aggregated Aβ is found in a fibrillar form: long and insoluble fibres bound together forming the amyloid plaques. The soluble Aβ is in the oligomeric form and recent evidence suggest it to be toxic.277,278 The role of fibrillar and oligomeric Aβ in the pathogenesis of AD remains unclear, however it is believed that polymerised Aβ, from small oligomers to large fibrils, is detrimental.279 Since the aggregated and soluble forms of Aβ occur particularly in AD patients, steps in the process from the membrane associated Aβ to the plaques are key events.  5.1.3 Metal Ion Involvement in Alzheimer’s Disease The brain tightly regulates metal ion concentrations as a part of normal physiological processes.  However, the metal ion concentration in the brain  increases as a result of normal aging.280,281  Various lines of evidence have  implicated metal ions, in particular Cu, Zn, and Fe in the pathogenesis of AD.282,283  106  There is evidence that normal enzyme-assisted homeostasis of these metal ions is drastically altered in AD brains.284  With increasing age, these homeostatic  mechanisms become less efficient and increases in metal ion concentrations are observed. Copper(II) is concentrated in Aβ plaques and is found in high concentrations up to 400 μM, almost double the normal brain extracellular concentration.285,286 It is thought that the Cu in the AD brain is miscompartmentalised rather than universally elevated as studies from bulk tissue indicate that total Cu concentration in an AD brain is comparable to the age matched controls.278,287 Similarly, zinc(II) is concentrated in Aβ plaques and can be found as high as approximately 1 mM.288 There is evidence for elevated Zn levels in AD tissues compared to control tissues in various brain regions and Zn levels in bulk tissues are more difficult to quantify.285,289 The third transition metal found localized in amyloid plaques is iron(III) in as high concentrations as 900 μM.288 The roles of metal ions in the aggregation of Aβ peptides and in ROS production are two key events that have been of particular interest in the pathophysiology of AD. Aβ is a metalloprotein that displays high affinity binding of Cu2+, Zn2+, and Fe3+.290 The metal binding site is found to be in the N-terminal region of the peptide and the process of Aβ misfolding and aggregation leading to plaques is greatly influenced by the aforementioned metal ions. Zn2+ has been indicated to have a primary role in inducing precipitation of Aβ along with its capability to build up protease-resistant Aβ aggregates.291 In vitro studies indicate  107  that Zn2+ rapidly precipitates soluble Aβ into amyloid aggregates292, and it is believed that the aggregation is promoted by intermolecular His(Nτ)-Zn2+-His(Nτ).293 Like Zn2+, Cu2+ acts as a potent mediator of Aβ aggregation under conditions of mild acidosis.294 Fe3+ also induces Aβ aggregation295, however, it does it to a lesser degree than does Cu2+.294 Transition metals such as Cu2+ and Fe3+ are capable of mediating the oxidative stress mechanism of Aβ toxicity. Aβ dissolved in Cu2+ or Fe3+ containing media is toxic to neurons, while Aβ in the absence of redoxactive metals is not toxic.296,297  There is good evidence that AβCu2+ or AβFe3+  complexes produce ROS, such as H2O2 which mediates toxicity.298,299 Furthermore, the addition of the H2O2 scavenging enzyme catalase to the cell cultures inhibits Aβ toxicity, which suggests that Aβ neurotoxicity is mediated by ROS generation.298,300 Co-incubation of Aβ with metal chelators has also shown to attenuate toxicity.300  Figure 5.3  Schematic view of the amyloid cascade hypothesis.301  108  The production of H2O2 is correlated to the early aggregation process,302 (Figure 5.3), which supports the amyloid cascade hypothesis that the early stages of aggregation are the toxic forms.303 The exact mechanism of ROS production is not known, but it is thought to occur through the reduction of Cu2+ and Fe3+, using O2 and biological reducing agents.298,299,304  A proposed reaction pathway for the  sequence of electron transfers that converts O2 into H2O2 is described in Scheme 5.1.282,301,304 An electron is initially transferred from Aβ peptide to reduce the metal •  ion forming a positively charged particle, Aβ+ . The reduced metal ion then reacts •  with O2 to form O2 - which is then converted to H2O2. After electron donation to O2, the radicalized Aβ peptide-metal complex might be restored by electron transfer from biological reducing agents such as Vitamin C, dopamine, and catecholamines.304 •  The generated H2O2 may degrade forming a highly reactive OH via Fenton chemistry or a Haber-Weiss reaction as outlined in Equation 5.1 and Equation 5.2, respectively.305,306 The biological implications of the ROS produced by Aβ result in protein oxidation, lipid peroxidation, cellular dysfunction and subsequent neuronal death.307  Equation 5.1 Fenton chemistry.  M n + + H2O2  → M ( n +1)+ + OH • + OH −  Equation 5.2 Haber-Weiss reaction.  O2− + H2O2  → OH • + OH − + O2  109  H2O2 + O2  O2 + 2H+  O2 AβM(n+1)+  Aβ Mn+  Aβ M(n+1)+  Reducing Substrates  Oxidized Products  Scheme 5.1  O2  Proposed mechanism of H2O2 production by Aβ bound to redox active metal.304  5.1.4 The Role of Met35 in Aβ Special attention has been paid to the role of Met35, which has been shown to play a critical role in the oxidative stress and neurotoxicity properties exhibited by Aβ. The generation of ROS by Aβ requires the reduction of metal ions Cu2+ or Fe3+, thus inducing oxidation of another moiety. The most likely candidate for oxidation is the sulfur atom of Met35.308  Met35 may serve as a source of electrons for the  reduction of molecular oxygen to H2O2.309-311 Consistent with this hypothesis is the evidence of oxidized Met35 that is found in post-mortem AD plaques.175,312 Additional evidence includes the mutation of the methionine to valine or norleucine,  110  which greatly reduces the rate of H2O2 generation313 even in the presence of other reducing agents.312 The redox potentials for Met oxidation and Cu2+ reduction result in a thermodynamically unfavourable electron transfer process.165 The peak potential for the Met35 oxidation to its radical cation has been reported to exceed 1.5V vs SHE in aqueous solution.314 The originally proposed redox potential for AβCu2+ was 0.75V vs SHE.298 Recently this redox potential was redetermined to be substantially less positive 0.28V vs SHE,315 rendering the redox reaction even less favourable. However, stabilization of the sulfur radical cation by amide moieties and the Cterminal carboxylate ion in Met peptides has been reported.167,310,316  The  neighbouring group participation in the oxidation of thioethers not only stabilizes, but may also render the oxidation potential of thioethers less positive, which will be easier to oxidize.317 For Aβ, modeling studies indicate that the amide carbonyl group of isoleucine at position 31 is in close proximity to the sulfur of Met35 and is proposed to stabilize the corresponding sulfur radical cation by bond formation.318  The  oxidation of Met35 causes rearrangement of the helical conformation present within the membrane-spanning region of Aβ.174 This rearrangement caused by the Met35 oxidation is proposed to be a cause for the elevation of soluble Aβ in Alzheimer’s disease,300 as the oxidized Aβ becomes less strongly associated with the membrane. To date there have been many inconsistencies in the literature regarding Met35 oxidation. Mass spectrometry has shown that Cu2+ ions are able to oxygenate Aβ with the most likely candidate being the sulfur atom of Met35.308,319 However,  111  recent mass spectrometry results have shown no indication of Met35 oxidation.315 Most of the research in identifying Met oxidation involves use of H2O2, which is known to oxidize Met, but a direct observation of metal-induced oxidation of Met35 is lacking. Thus, this chapter focuses on the work of metal-induced oxidation of Met only in the presence of redox active metals. Since Sulfur K-edge XAS is an element specific technique, it is an ideal spectroscopic tool to investigate the oxidation of a single sulfur containing amino acid, Met35 in Aβ in the presence of metal ions such as Cu2+ and Fe3+. The aim of this study is to test if the sulfur atoms in Met35 in Aβ or Met in N-acetyl-L-methionine amide are altered in any way in the presence of redox active Cu2+ and Fe3+ in the absence of exogenous reducing agents and H2O2.  5.2  Experimental  5.2.1 Materials Synthetic Aβ1-40 peptide was obtained from Bachem (Torrance, CA, USA). Aβ1-16 was synthesised on a CS Bio Co peptide synthesizer using standard Fmoc chemistries and subsequently purified by high performance liquid chromatography (HPLC) as previously described.320 The synthesis and purification was carried out by Dr. Jinhe Pan (past graduate student from Dr. Straus’ lab in UBC). The purified products were confirmed by mass spectral analysis. Both Aβ1-40 and Aβ1-16 were stored at -20oC until use. Water of ultrahigh quality (TraceSELECT® Ultra) was purchased from Sigma-Aldrich. CuCl2, FeCl3, and phosphate buffered saline (PBS) tablets were also purchased from Sigma-Aldrich.  N-acetyl-L-methionine amide  (purity 99%) was purchased from Bachem.  112  5.2.2 Sample Preparation Aβ1-40 (1 mg) was dissolved in 200 μL of 50 mM PBS buffer, pH 6.5 with 40% glycerol (v/v). Inclusion of glycerol as a cryoprotectant for biological samples is an accepted method for soft XAS data collection.  To this peptide solution, metal  solution (CuCl2 or FeCl3) was added so that the peptide to metal ratio was 1:1. The final concentration of Aβ1-40 in the peptide-metal mixture was approximated to 1 mM. The stock metal solutions were also made in 50 mM PBS buffer, pH 6.5 with 40% glycerol (v/v). N-acetyl-L-methionine amide (ac-Met-NH2) samples were prepared in similar manner to Aβ1-40, but with higher concentrations of approximately 5 mM.  Metal  solutions were also added to ac-Met-NH2 solutions so that ratio of ac-Met-NH2 to metal was 1:1.  Additional samples were also prepared with ac-Met-NH2 which  included the presence of Aβ1-16 to mimic the metal binding mode of Aβ peptide. For samples with H2O2, 3% H2O2 (v/v) was added to metal solutions or was directly added to Aβ1-40 or ac-Met-NH2 solutions. All the reactions were carried out in the absence of light to avoid photochemical reactions and were incubated at 37oC for a period of 1-2 hours. The buffer and metal solutions were tested for any sulfur contamination with S K-edge XAS prior to addition to the samples.  113  5.2.3 XAS Data Acquisition XAS data were collected at SSRL on beamline 6-2 under conditions described in section 3.2.8.  5.2.4 Data Processing and Analysis Complete XAS data reduction and analysis is described in section 2.3.1.  5.3  Results and Discussion  5.3.1 Copper(II)-Induced Oxidation Cu2+-induced oxidation of Met was investigated by repeating the experiments from previously published evidence.308 This required the Aβ1-40 to be incubated with Cu2+ in the presence of H2O2. The sulfur contribution in Aβ1-40 came from a single sulfur atom from Met35, thus the pre-edge feature at 2473.5 eV in the S K-edge XAS spectrum Aβ1-40 was due to the sulfur of Met35, (Figure 5.4). In agreement with the literature findings, this sulfur atom in the Cu2+/H2O2 system was modified to give a pre-edge feature at 2476.3 eV, corresponding to methionine sulfoxide (MetSO). Since H2O2 has strong oxidizing properties and is known to oxidize Met to MetSO, a reaction of Aβ1-40 only in the presence of H2O2 without Cu2+ was also performed. As expected, the same pre-edge feature at 2476.3 eV was observed, suggesting direct peroxide oxidation of Met35 to MetSO (Figure 5.4). Since H2O2 alone was sufficient to oxidize Met35, this brings into question the role that Cu2+ plays in the oxidation of Met35 in the Cu2+/H2O2system. It seems that H2O2 is at least partly responsible for  114  the observed Met oxidation of Aβ peptide in the Cu2+/H2O2 system. Furthermore, to investigate whether Aβ1-40 Met35 oxidation was unique to the peptide, similar reactions using N-acetyl-L-methionine amide (ac-Met-NH2) were performed. Both, in the Cu2+/H2O2 system and in H2O2, ac-Met-NH2 was oxidized to MetSO indicating that the observed oxidation of Met in Aβ1-40 was not specific to the peptide alone, but to Met in general.  Figure 5.4  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Cu2+/H2O2 or H2O2.  To investigate whether Cu2+ alone can induce oxidation of Met35, a reaction involving Aβ1-40 in the presence of Cu2+ only was used. Figure 5.5 shows evidence of Met35 oxidation to MetSO in Aβ1-40 in the presence of Cu2+ under aerobic  115  conditions. Met is thought to be a possible electron source for the reduction of •  •  bound Cu2+, which would then result in Cu1+ and MetS +. The generated MetS + can react with O2 to form MetSO (see section 4.1.3). Another possibility of Met oxidation •  might be that the reduced Cu1+ is able to transfer an electron to O2 generating O2 (see Scheme 5.1), which can then undergo fast reactions with MetS•+ producing the observed MetSO.  Figure 5.5  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Cu2+.  In order to substantiate that this Cu2+-induced oxidation is unique to Aβ peptide, ac-Met-NH2 was also subjected to a reaction with Cu2+.  This reaction  however, failed to produce the pre-edge feature at 2476.3 eV that corresponds to  116  the formation of MetSO, which suggests that perhaps binding of Cu2+ to the Aβ peptide is necessary for the observed Met35 oxidation in Aβ. Thus, to mimic the metal binding mode in Aβ1-40, a shorter fragment of the peptide, Aβ1-16 was used since the metal binding region is thought to be in the N-terminal region. Therefore, a reaction with ac-Met-NH2 in the presence of Aβ1-16 bound to Cu2+ was tested and this also failed to produce the MetSO oxidized peak, (Figure 5.6). The absence of MetSO formation in ac-Met-NH2 with Aβ1-16Cu2+ was consistent with finding that Cu2+ was not reduced with the addition of methionine to Aβ1-16 or Aβ1-20.321,322 It seems as though the Cu2+-induced oxidation of Met is only observable in the full-length Aβ peptide, which might be due to reduction of the bound Cu2+. The lack of MetSO production in Aβ1-16Cu2+ with ac-Met-NH2 also suggests that H2O2 might not have been produced, as it had been shown earlier that the presence of H2O2 alone can oxidize Met.  Recent findings suggest that, in the  absence of reducing substrates, production of H2O2 is not observed in the Aβ1-40Cu2+ and Aβ1-16Cu2+. However, the reduction of the bound Cu2+ persists in Aβ1-40 while Aβ1-16 with an exogenous Met source still failed to reduce the bound Cu2+.322 This is consistent with the production of MetSO in full-length Aβ, which suggests that the bound Cu2+ must be reduced to Cu+ but the source of the electron may not be Met35. •  •  Once Cu+ is formed, it may then generate O2 - via electron transfer to O2. The O2 being highly reactive and is good oxidizing agent, it can react with Met35 to generate the observed MetSO.  Furthermore, the lack of MetSO formation in Aβ1-16Cu2+  suggests that ac-Met-NH2 does not provide the electron for the Cu2+ reaction, thus it •  is unable to generate O2 - which can then react with ac-Met-NH2.  As a result, the  117  source of the electron for Cu2+ must come from the Aβ peptide since external reducing substrates were not available in the reaction. This also suggests that the electron source might be the missing residues 17-40 which lack in Aβ1-16. Another possibility is that Met35 still might be the electron source for the Cu2+ reduction but Met might be too remote from the metal centre for direct electron transfer.238 Thus, it requires intermediate residues between the Met and the metal centre to mediate electron transfer though the peptide from Met to the metal centre. This possibility has been proposed from computational studies which suggest that phenylalanine at position 20 might play such role.323  Figure 5.6  S K-edge XAS spectra of N-acetyl-methionine amide (ac-Met-NH2) before and after incubation with Aβ1-16Cu2+.  118  5.3.2 Iron(III)-Induced Oxidation The result of Aβ1-40 in a Fe3+/H2O2 system is shown in Figure 5.7 along with Aβ1-40 with H2O2. As expected, Met35 is oxidized to MetSO as evidenced by the preedge feature at 2476.3 eV, and H2O2 is the oxidizing agent that is responsible for the Met35 oxidation. The result was the same when the source of Met is changed to acMet-NH2. In the absence of H2O2, the Fe3+ did not oxidize Met35 in Aβ1-40, (Figure 5.8). This was a little surprising since Cu2+ alone caused an oxidation of Met35, but Fe3+ failed to do so. This raises questions of whether Fe3+ was binding to Aβ: if so, is the binding mode similar to that of Cu2+?  There is limited evidence in the  literature regarding Fe3+ interaction with Aβ. Most of the research has been focused on Cu2+ and the assumption is that since both Cu2+ and Fe3+ are redox-active, they should have similar redox chemistry with Aβ.  This assumption has not been  investigated to this point. The differences in the oxidation of Met35 may be attributed to whether the Fe3+ binds to the Aβ or not and whether the binding modes are similar between the Cu2+ and Fe3+. In order to get a complete understanding of these differences the metal-binding regions must be probed which may give insight into this. This will be pursued in Chapter 6. ac-Met-NH2 was subjected to the same reaction as Aβ, which failed to produce any oxidized Met in Fe3+ solutions under aerobic conditions. Furthermore, Aβ was also mimicked by using the shorter fragment of the peptide Aβ1-16. The Fe3+ bound to Aβ1-16 still failed to oxidize ac-Met-NH2.  119  Figure 5.7  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Fe3+/H2O2 or H2O2.  Figure 5.8  S K-edge XAS spectra of Aβ1-40 peptide before and after incubation with Fe3+.  120  5.4  Conclusions Much of the research to date has focused on the redox chemistry of Cu2+ in  Aβ peptide and has produced inconsistent findings in regards to the role of Met35 and the oxidation state of Met35. The inconsistencies are likely largely due to the use of H2O2 to observe the metal-induced oxidation and the lack of direct detection methods to observe the changes in the Met35 residue. Sulfur K-edge XAS is an ideal spectroscopic tool to detect the changes in the Met residue since Aβ peptide has a single sulfur atom in the entire peptide in Met35.  The full-length peptide in the  presence of Cu2+ contained an oxidized species in Met35. When Cu2+ was bound to the Aβ1-40, there was evidence of MetSO formation, which is consistent with the finding of post-mortem AD plaques. Mimicking the metal binding region of Aβ and adding an exogenous Met is not sufficient to oxidize the Met, which suggests that the observed Met oxidation is unique to the full-length peptide. Surprisingly, Fe3+ does not oxidize the Met residue in the full-length peptide. Until now the redox chemistry of Fe3+ has been thought to be similar to Cu2+. Here I report that the Aβ peptide redox chemistry is different between the two metal ions. The next step in understanding the difference in the effect of the two metals on Aβ peptide is to investigate whether the two metals bind in similar fashion.  121  CHAPTER 6: STRUCTURAL CHARACTERIZATION OF THE COPPER(II) AND IRON(III) COORDINATION TO THE AMYLOID-BETA PEPTIDE  6.1  Introduction  6.1.1 Experimental Techniques Metal ions such as Zn2+, Cu2+, and Fe3+ bind to the N-terminal region of Aβ peptide. Crystal structure analysis using X-ray diffraction is, in many cases, the most effective method available for obtaining high-resolution structural information about metalloproteins.  Neither Aβ1-40 nor Aβ1-42 crystallize due to peptide  aggregation in the presence of metal, so crystallographic data are not available. Thus, many alternative structural methods have been used during the last decade to try to characterize the structure of the metal binding site in Aβ complexes, including EPR,324,325 NMR,325,326 Raman spectroscopy,175,293 and XAS.327-329 XAS, provides structural information about the absorber atomic environment through the study of the oscillations of the absorption coefficient that originates from the atoms surrounding it. This interference spectrum (EXAFS) contains detailed information about scatterer-absorber relative positions, from which the nature and geometric arrangement of the amino acids that are coordinated to the metal can be inferred with good accuracy.  122  6.1.2 Aβ-Cu Complexes The coordination chemistry of Cu2+ has been studied in the past few years with truncated Aβ1-16 and Aβ1-28 and with native Aβ1-40 and Aβ1-42 peptides; however, no real consensus has been reached regarding the identity of the coordinating ligands. Earlier EXAFS measurements of AβCu2+ led to a model in which the Cu2+ ion binds to Aβ in a five-coordinate square-pyramidal ligand environment.327 This coordination environment involves ligation of three imidazoles from the His at positions 6, 13, and 14, an oxygen atom donor from Tyr at position 10 and an axial water molecule. However, other EXAFS studies have been used as evidence for either a distorted six-coordinate329 or four-coordinate330 Cu2+ binding site in Aβ peptide. The six-coordinate model329 involves the three imidazoles of His6, His13 and His14 as well as a carboxylate oxygen from either glutamic acid (Glu11) or aspartic acid (Asp1) in an approximately equatorial planar arrangement, with the axial ligands consisting of a water molecule and the other carboxylate oxygen also from Glu11 or Asp1. The four-coordinate model330 proposes involvement of two His residues and two nitrogen and/or oxygen ligands. Both the six-coordinate and four-coordinate models agree on the absence of Tyr from the coordination environment. Furthermore, other spectroscopic data such as EPR324 and theoretical work have also yielded conflicting conclusions with regards to the identity of the ligands as well as the coordination number.325,331  Recent pH-dependence studies of Cu2+  coordination to Aβ peptide revealed that two complexes, distinguishable by conventional 9 GHz EPR, are present near physiological pH.332 These complexes are called component I (lower pH values) and component II (higher pH values), both  123  of which having a five-coordinate geometry and the absence of Tyr as a coordinating ligand. There is still significant controversy regarding the physiologically-relevant form of Cu2+-bound Aβ peptide.  6.1.3 Aβ-Zn Complexes In the case of the Aβ-Zn2+ complex, the situation appears to be even more complicated and several coordination modes have been proposed.  NMR  investigations suggests an intermolecular dimer of Aβ, with Zn2+ binding involving several His and the peptide N-terminus.326 Further support for this unusual binding mode was obtained from an XAS study of Aβ13-21 bound to Zn2+ in which pairs of peptides appear to be cross-linked by a Zn2+ bridge binding two His residues, one from each of the peptides.333  Further NMR studies propose intrapeptide  coordination involving three His residues (His6, His13, and His14) and the Nterminus.334,335 Opposing this viewpoint, another Zn K-edge XAS study suggests a four His residue coordination mode.328 Given that a fourth His residue is lacking in the Aβ peptide sequence, this proposal would require a Zn2+ bridged interpeptide coordination.  6.1.4 Aβ-Fe Complexes The redox-active metal ion Fe3+ is found in AD plaques, suggesting that it might mediate ROS generation. Thus, answers to fundamental questions regarding the coordination environment and reactivity of Fe3+ when bound to Aβ peptide are essential; however, much of the investigation on the metal binding of Aβ peptide has  124  been focused on Cu2+ and Zn2+. It is generally assumed that Fe3+ has a similar binding mode as Cu2+ and has similar reactivity as a result. Recent evidence of Fe3+ binding to Aβ peptide comes from electrospray ionization mass spectrometry (ESIMS),336 however, this study did not reveal any details on the coordination environment of Fe3+. In order to get a better understanding of the differences of the metal-induced Met35 oxidation that was observed with Cu2+ and Fe3+ in the full-length peptide Aβ (Chapter 5), the metal binding site of Cu2+ and Fe3+ must be investigated. Thus, this chapter looks at the metal binding site of Cu2+ and Fe3+ in the full-length Aβ peptide as well as the truncated Aβ1-16.  6.2  Experimental  6.2.1 Materials Materials used for this study are described in section 5.2.1.  6.2.2 Sample Preparation Aβ1-40 (1 mg) was dissolved in 200 μL of 50 mM PBS buffer, 75 mM NaCl, pH 6.5 with 40% glycerol (v/v) as a cryoprotectant. To this peptide solution, the metal solution (CuCl2 or FeCl3) was added so that the peptide to metal ratio was 1:1. The final concentration of Aβ1-40 in the peptide-metal mixture was ~1 mM. The stock metal solutions were also made in 50 mM PBS buffer, 75 mM NaCl, pH 6.5 with 40% glycerol (v/v). Immediately after preparation, the samples were injected into XAS  125  cells sealed with a polypropylene window on the front, and rapidly frozen in liquid nitrogen. Aβ1-16 samples were prepared in similar manner to Aβ1-40, but the final peptide concentration was ~3 mM.  6.2.3 XAS Data Acquisition Fe K-edge XAS and Cu K-edge XAS data were collected at the SSRL on beamline 7-3 under ring conditions of 80-100 mA at 3.0 GeV. This setup used a 20pole, 2 Tesla wiggler, 0.8 mrad beam and a Si (220) double-crystal monochromator. The beamline is equipped with harmonic rejection mirrors which can efficiently minimise higher harmonic contamination. The incident X-ray intensity (I0), sample absorption (I1), and Fe or Cu reference (I2) were measured as transmittance in three consecutive N2-filled ionisation chambers and fluorescence data were collected using a 30-element Ge detector array. The fluorescence signal was windowed at Kalpha emission of the element, either Cu or Fe depending on the sample. For each sample, 10 to 15 sweeps were taken and all data were measured to k = 15 Å-1. All samples were maintained at 15 ± 3 K throughout data collection by using a continuous-flow liquid helium cryostat. Photoreduction of Cu2+ or Fe3+ by the X-ray beam was monitored by comparing edge spectra (XANES) for consecutive sweeps. All the experiments were repeated to ensure reproducibility.  6.2.4 Data Processing and Analysis ATHENA software221 was used for Cu and Fe K-edge XANES data reduction. For energy calibration, the lowest-energy maximum of the first derivative of the  126  reference spectra was assigned, for the Cu K-edge was set to 8979 eV and for Fe K-edge set to 7112 eV. Data scans were averaged and background subtraction and normalization were performed simultaneously using a linear pre-edge and postedge function. The averaged energy calibrated data were used for EXAFS analysis. The EXAFS oscillations χ(k) were extracted using an automated background subtraction AUTOBK algorithm337 implemented in the ATHENA software.  The EXAFS  oscillations χ(k) were quantitatively analyzed by the ARTEMIS software, using ab initio theoretical amplitude, phase, and mean-free path factors calculated by FEFF6.  FEFF is an automated program for ab initio multiple scattering (MS)  calculations of EXAFS. The atomic coordinates (x, y, z) for the theoretical model were generated by a software program based on MATLab that has been developed by Dr. Mario Jaime-Delgado (past graduate student in Kennepohl lab). The atomic coordinates were used in the FEFF6 input file to generate the multiple scattering paths.  6.2.5 Model Building and Fitting Strategy To get reliable information from EXAFS data, a crucial point of analysis is the choice of the initial trial geometry around the absorber. Lacking crystallographic data for Aβ peptides, the only indication about possible metal ligands comes from the knowledge of the peptide amino acid sequence. Also, previous spectroscopic evidence of ligand coordination is helpful in building a theoretical model.  127  Initial fits of the EXAFS data were performed with only a single scattering (SS) shell. This simple scattering model gives reasonable fits, but cannot account for scattering waves corresponding to longer distances and is insufficient for developing an accurate representation of metal-binding. More detailed models, including multiple scattering, were investigated with many of the suggested models from the literature.324,325,327,329,332 In both, Cu2+ and Fe3+ EXAFS, the starting models were remodelled accordingly to give good fit in the R-space. Constrained and restrained refinement procedures338 were followed, in which the His imidazole and the Tyr phenyl rings are treated as rigid bodies. Constrained and restrained refinement minimizes the number of free parameters in the leastsquares refinement to increase the degree of determinacy of the model.  Light  scatterers such as nitrogen and oxygen atoms are difficult to distinguish based solely on their individual contribution to the EXAFS signal; however, when these atoms are tightly anchored to a large and well ordered structure such as an imidazole or a phenyl ring, they can be unambiguously identified due to the multiple scattering contributions, in general. The structural parameters refined during constrained/restrained refinements included the distance ri of ligands and the Debye-Waller factors σi2 for each ligand. The Debye-Waller factors for atoms at similar distances for a given ligand type were grouped together.  In addition to the Debye-Waller factor for the atoms directly  coordinated to the metal, only the Debye-Waller factor for the second and third shell atoms of the rigid ligands were selected for refinement. The remaining terms were fixed at a value of 0.010 Å2. The coordination number Ni for each type of atom was  128  kept fixed.  In addition to the structural parameters, the photoelectron energy  threshold (ΔE0), which was treated as a single overall parameter for the multiple shell fits, was varied to correct for the simple estimate of E0 made in ATHENA. The amplitude reduction factor S02 was allowed to be adjusted around the starting value 0.9. The χ(k) data were weighted by k3 and windowed between 2 < k < 12 Å-1 using a Kaiser-Bessel window with dk = 2.0 Å-1 to minimize spectral ringing. The fits were carried out in χ(r) in the region of 1 < r < 5 Å using a Kaiser-Bessel window with dr = 0.2 Å. All SS contributions of atoms ≤ 5 Å and all MS contributions > 10% and l ≤ 4 (triple scattering paths with four legs) were included in fits. Best fits to the experimental data were determined by selecting the model that gave both chemically reasonable refinement parameters and the lowest R-factor automatically calculated by ARTEMIS.  6.3  Results and Discussion  6.3.1 AβCu2+ Complexes The Cu K-edge XAS data obtained for the Cu2+ bound Aβ1-40 and Aβ1-16 are shown in Figure 6.1 along with Cu2+ in 50 mM PBS buffer. In both peptides, the preedge is characterized by a weak transition at 8979.7 eV that corresponds to the electric quadrupole allowed Cu 3d ← 1s transition (labeled a in Figure 6.1) and a shoulder in the pre-edge region at 8986.8 eV that corresponds to a Cu 4p ← 1s transition with a metal-ligand charge-transfer shakedown (labeled b in Figure 6.1).339 The corresponding normal Cu 4p ← 1s transition is not resolvable from the edge.  129  Figure 6.1  The XANES region of the Cu K-edge XAS of AβCu2+. The pre-edge transition of 3d ← 1s is marked a and b represents the 4p ← 1s + shakedown transition.  Figures 6.2A and B show the EXAFS region of Aβ1-16Cu2+ and Aβ1-40Cu2+, respectively. The similarity in the XANES and EXAFS regions between Aβ1-40 and Aβ1-16 suggests that geometry of the Cu2+ coordination does not change significantly. In both peptides, initial fits of the EXAFS data with only a single-scattering (SS) shell including N/O scatterers suggest a coordination number (CN) of 5 for Cu2+. Fourier-transformed EXAFS data in R-space (Figures 6.2C and D) shows strong outer-sphere scattering between r = 2 and 4 Å, which is characteristic of MS contributions between the Cu centre and rigid ligands such as imidazole rings from  130  His residues. Using a MS analysis, the number of imidazole ligands was determined to be three in the coordination environment. Addition of more or fewer imidazole ligands yielded poorer fits to the data. This preference of Cu2+ binding to nitrogen of imidazole is in accordance with the principle of hard/soft acids and bases. For the remaining two ligands, various models were tested. Based on the previous spectroscopic evidence, the candidates included the phenolate group from the Tyr residue and a water molecule or carboxylate group of glutamic acid (Glu) or aspartic acid (Asp). A model involving three imidazoles of His and two oxygen of the carboxylate group either from Glu or Asp residue gave a good fit for the Cu2+ coordinated in Aβ1-40 and Aβ1-16 (Figure 6.2, Table 6.1). Available His residues in Aβ peptide are His6, His13 and His14 account for the three N(His) contributions in the EXAFS fit. The two oxygen atoms are presumed to be from the N-terminal Asp1.340,341 However, Asp at position 7 and Glu at position 3 and 11 are also potential carboxylate donors. The strongest argument for Asp1 as the coordinating ligand comes from the finding that the mutation of Asp1 to asparagine (Asn) affects the EPR spectrum.340 Use of oxygen from phenolate and a water molecule yielded a poorer fit to the data; this is also consistent with UV-Vis spectroscopic data. Cu2+-phenolate containing complexes exhibit a characteristic intense charge transfer band around 400 – 500 nm, but such a band has never been observed in this system.320,342,343 In addition, NMR320,325 and EPR325,332,344 studies are in agreement with Tyr not being involved in the coordination geometry. Furthermore, the pKa of Tyr10 has been determined320 to be identical to that of a free Tyr residue (pKa ~ 10.1), which suggests that Cu2+ is not bound to the Tyr residue.  131  Copper binding is in competition with protonation, which means that Cu2+ binds to N/O ligands with a low pKa, which favours a carboxylate group of Asp (pKa ~ 3.8) and His (pKa ~ 6.5). Also, the involvement of a water molecule as a ligand was rejected based on the EPR study of  17  O-labeled H2O.344 Thus the most likely model  is that of Cu2+ bound to three His and the carboxylate group of Asp. A schematic picture of the molecular arrangement is shown in Figure 6.3.  Table 6.1  EXAFS parameters for Aβ1-16Cu2+ and Aβ1-40Cu2+.  Sample *  AβCu2+  AβCu2+  AβCu2+  Cu2+ (buffer)  S02  (Å )  ΔE0 (eV)  Rfactor  1.95  0.0039  0.93  0.88  0.0177  2  1.99  0.0051  N (His)  2  1.94  0.0031  1.21  0.94  0.0416  N/O  2  2.04  0.0040  N (His)  3  1.95  0.0039  1.02  0.89  0.0279  O (Glu/Asp)  2  1.99  0.0050  O (H2O)  1  2.03  0.0054  O (H2O)  4  1.97  0.0032  1.98  0.90  0.0091  O (H2O)  2  2.36  0.0081  Coordinating Ligand  N  N (His)  3  O (Glu/Asp)  R (Å)  σ2 2  * Since Aβ1-16Cu2+ and Aβ1-40Cu2+ data are superimposable, their fits yielded the same EXAFS parameters.  N = path degeneracy, R = distance in Å, σ2 = Debye-Waller factor, S02 = amplitude reduction parameter, ΔE0 = internal reference energy. The R-factor indicates quality of fit and is calculated in R-space. For AβCu2+ (k3, Δk = 2 – 12 Å-1, Δr = 1 – 5 Å), for Cu2+ in buffer (k3, Δk = 2 – 12 Å-1, Δr = 1 2.5 Å).  132  Figure 6.2  EXAFS of Aβ1-16Cu2+and Aβ1-40Cu2+. A) and B) are k3 weighted χ(k) of Aβ12+ and Aβ1-40Cu2+, respectively. C) and D) are corresponding Fourier16Cu transformed (FT) of Aβ1-16Cu2+ and Aβ1-40Cu2+. Experimental data are shown by dashed lines and simulations to the data by solid lines.  133  Figure 6.3  Schematic picture of the molecular arrangement around the Cu2+ in Aβ. Atom colours are as follows: copper (Cu) black; oxygen (O) red; nitrogen (N) blue; carbon (C) gray.  Previous EXAFS studies indicate a four-coordinate330 and a six-coordinate329 Cu2+ binding to Aβ peptide. The four-coordinate model suggests Cu2+ coordinated to two imidazole of His residue and two nitrogen/oxygen ligands, while the sixcoordinate model is similar to the proposed five-coordinate model (Figure 6.3) with an additional water molecule. The corresponding four-coordinate and six-coordinate model fits are shown in Figure 6.4 and EXAFS parameters in Table 6.1. These models yield poorer fits to the data, thus strengthening the conclusion that the fivecoordinate model is most reasonable for Cu2+ binding in Aβ peptide at pH 6.5.  134  Figure 6.4  Four-coordinate and six-coordinate fits of Aβ1-40Cu2+. A) and B) are k3 weighted χ(k) of Aβ1-40 with four-coordinate and six-coordinate Cu2+ fits, respectively. C) and D) are corresponding fits in R-space. Experimental data are shown by dashed lines and simulations to the data by solid lines.  135  The inconsistency with previous EXAFS studies is likely due to the dynamic nature of Aβ and the existence of multiple forms at physiological pH.332,342  All  previous EXAFS studies were done at pH 7.2 to 7.4, thus the available data are for mixtures of two forms of AβCu2+.  According to EPR332 and potentiometric  studies,320,340,342 at pH 6.5, only component 1 is present, thus a single species should be observed. Furthermore, only one EXAFS study327 proposed Tyr as a coordinating ligand, however, a high R-factor of 32% for this simulation is reported even though they were calculated in k-space, which includes high frequency noise components. For completeness, the parameters of the best fit of the Cu2+ buffer sample is reported in Table 6.1 and a single-scattering (SS) fit is shown in Figure 6.5. In the absence of peptide, Cu2+ is in a six-coordinate geometry with oxygen atoms, probably from water molecules. Also, multiple scattering features observed for the Cu2+ bound Aβ peptide at 2 to 4 Å are not observed in the peptide-free Cu2+. The EXAFS analysis of Cu2+ buffer sample corresponds with others.345,346  136  Figure 6.5  EXAFS of Cu2+ in buffer. A) is k3 weighted χ(k) of Cu2+ in buffer and B) is the corresponding Fourier-transformed (FT) of Cu2+ in buffer Experimental data are shown by dashed lines and simulations to the data by solid lines.  6.3.2 AβFe3+ Complexes The XANES region of Fe3+ bound Aβ1-40 and Aβ1-16 is depicted in Figure 6.6 along with the Fe3+ in 50 mM PBS buffer. In both the peptides and the Fe3+ in buffer, the pre-edge is characterized by a weak transition at 7114.5 eV that corresponds to  137  the electric quadrupole allowed transition Fe 3d ← 1s. This pre-edge feature is split by ~ 1 eV (inset of Figure 6.6). This pre-edge feature has shown to be sensitive to the geometry of the iron atom and the splitting of the pre-edge feature has been attributed to an octahedral geometry of the iron atom.347 The Fe K-edge EXAFS region of Fe3+ bound to Aβ1-16 and Aβ1-40 is shown in Figures 6.7A and B. As seen with Cu2+ coordination, the Fe3+ XANES and EXAFS spectra are similar for both the full-length and the truncated Aβ peptide. In both peptides, the single-scattering analysis has shown that Fe3+ is in a six-coordinate environment.  Fourier-transformed EXAFS in R-space (Figures 6.7C and D) shows  outer-sphere scattering between r = 2 and 4 Å, but these are not as prominent as the ones found for Cu2+. Nevertheless, MS analysis suggests that two His residues are bound to the Fe3+ ion. The remaining four ligands are speculated to be carboxylate oxygen or a water molecule. Fe3+ preferentially binds to oxygen ligands, thus water molecules and carboxylate are the likely candidates.  The octahedral geometry  observed in EXAFS analysis is consistent with the observed splitting of the pre-edge feature in XANES confirming that Fe3+ is bound to Aβ in a six-coordinate environment. The schematic picture of the molecular arrangement of Fe3+ is shown in Figure 6.8.  An EXAFS fit for a five-coordinate geometry in which Fe3+ is  coordinated to two His residues and 3 nitrogen/oxygen ligands is also shown in Figure 6.9 and EXAFS parameters in Table 6.2. This further supports the proposed six-coordinate model.  138  Figure 6.6  The XANES region of the Fe K-edge XAS of AβFe3+. Inset shows the Fe 3d ← 1s pre-edge features.  For completeness, the Fe K-edge XAS of Fe3+ in the absence of Aβ peptide was also subjected to EXAFS analysis, (Figure 6.10). For Fe3+ in buffer, the best fit parameters are compatible with an octahedral geometry and the EXAFS parameters are reported in Table 6.2.347  139  Figure 6.7  EXAFS of Aβ1-16Fe3+ and Aβ1-40Fe3+. A) and B) are k3 weighted χ(k) of Aβ13+ and Aβ1-40Fe3+, respectively. C) and D) are corresponding Fourier 16Fe transformed (FT) of Aβ1-16Fe3+ and Aβ1-40Fe3+. Experimental data are shown by dashed lines and simulations to the data by solid lines.  140  Table 6.2  EXAFS parameters for Aβ1-16Fe3+ and Aβ1-40Fe3+.  Sample  Coordinating Ligand  N  R (Å)  σ2 (Å2)  ΔE0 (eV)  S02  Rfactor  *AβFe3+  N (His)  2  1.95  0.0021  - 1.37  0.92  0.0183  N/O  4  2.04  0.0063  N (His)  2  1.95  0.0021  -1.66  0.94  0.0205  N/O  3  2.04  0.0062  O (H2O)  4  1.94  0.0031  1.22  0.78  0.0003  O (H2O)  2  2.07  0.0036  AβFe3+  Fe3+ (buffer)  * Since Aβ1-16Fe3+ and Aβ1-40Fe3+ data are superimposable, their fits yielded same EXAFS parameters. N = path degeneracy, R = distance in Å, σ2 = Debye-Waller factor, S02 = amplitude reduction parameter, ΔE0 = internal reference energy. The R-factor indicates quality of fit and is calculated in R-space. For AβFe3+ (k3, Δk = 2 – 12 Å-1, Δr = 1 – 5 Å), for Fe3+ in buffer (k3, Δk = 2 – 12 Å-1, Δr = 1 2.5 Å).  Figure 6.8  Schematic picture of the molecular arrangement around the Fe3+ in Aβ. Atom colours are as follows: iron (Fe) black; oxygen (O) red; nitrogen (N) blue; carbon (C) gray.  141  Figure 6.9  Five-coordinate fit of Aβ1-40Fe3+. A) k3 weighted χ(k) of Aβ1-40 with fivecoordinate Fe3+ fit. B) is corresponding fit in R-space. Experimental data are shown by dashed lines and simulations to the data by solid lines.  142  Figure 6.10  6.4  EXAFS of Fe3+ in buffer. A) is k3 weighted χ(k) of Fe3+ in buffer and B) is the corresponding Fourier-transformed (FT) of Fe3+ in buffer Experimental data are shown by dashed lines and simulations to the data by solid lines.  Conclusions The metal-binding affinity of Aβ peptide is an important parameter because it  gives information about the biological significance of the metal binding. It is also important for therapeutic approaches in Alzheimer’s disease based on the withdrawal of metal ions from Aβ, but not from essential metal proteins.348  143  Although several spectroscopic techniques have been used to understand the metal binding site of Aβ peptide, much of the attention has been focussed on the redox-active Cu2+ and redox-inactive Zn2+.  No consensus has been reached in  regards to the coordination number or the identity of the ligands for Cu2+. The lack of consensus is most likely due to the dynamic nature of Aβ, which is sensitive to pH. The previous spectroscopic studies were done at physiological pH, at which two Aβ binding modes exits, component I (low pH) and component II (high pH).  The  EXAFS analysis of the Cu2+ bound Aβ presented here was performed at pH 6.5, where component I is the predominant species. The Cu2+ bound to Aβ has a fivecoordinate geometry with three His residues and carboxylate oxygen of Asp. This geometry is similar to the proposed six-coordinate geometry for AβCu2+ without the axial water molecule.329 Evidence of Fe3+ interaction with the Aβ in the literature is limited. It was assumed that since Fe3+ is a redox-active metal like Cu2+, it would have a similar binding mode and redox-active chemistry to Cu2+. Here I report that the coordination of Aβ differs between the two metals. While Cu2+ is shown to be in a five-coordinate geometry with a His-rich coordinating environment, Fe3+ is in a six-coordinate geometry with an oxygen-rich environment. This difference in binding may have some effect on the observed Met35 oxidation differences in Chapter 5. The lack of information on the Fe3+ bound Aβ peptide leads me to speculate on the identity of the remaining 4 N/O coordinating ligands.  Furthermore, the essential coordinating  ligands for Cu2+ and Fe3+ are in the N-terminal region of the peptide because the metal bound Aβ1-16 and Aβ1-40 produced superimposable EXAFS oscillations.  144  CHAPTER 7: CONCLUDING REMARKS AND FUTURE DIRECTIONS  Sulfur K-edge X-ray absorption spectroscopy has been used to investigate cysteine and methionine oxidation as it provides a direct spectroscopic evidence for the redox state of the sulfur atom in these amino acids. Through the findings from these specific projects in this thesis, a clear understanding was gained of the fundamental redox chemistries of Cys and Met and its implications in diseases such as age-related cataracts and Alzheimer’s disease. Although cysteine oxidation to the cysteinyl radical has been implicated in various biological functions, the applicability of the predominant spectroscopic method, EPR is limited.  These  challenges of detecting sulfur-based radicals with EPR led to the development of S K-edge XAS as a spectroscopic tool to evaluate the sulfur-based radical species. My work has been successful in investigating cysteinyl radicals in a protein system with S K-edge XAS. The cysteinyl radical is characterized by a pre-edge feature that corresponds to the 3p ← 1s transition, which is well separated from the near-edge feature of the sulfur. S K-edge XAS has also shown to be a sensitive method to detect these cysteinyl radicals in hydrophobic and hydrophilic protein environments. The pre-edge feature of the cysteinyl radicals that were in a hydrophobic environment was found to be lower in energy than that of cysteinyl radicals that were in a hydrophilic environment probably due to the hydrogen bonding interactions.  145  The usefulness of S K-edge XAS can further be investigated in detecting thiyl peroxyl radical (RSOO•), which have been shown to be very reactive and to have an important role in biology. Thus, the generated cysteinyl radical in the hydrophobic and hydrophilic environments of azurin can be probed with O2 to generate such radicals and see its interaction in these different environments. Furthermore, these cysteinyl radicals can also be probed with other biologically relevant small molecules such as NO, to understand the fundamental reactivity of these radicals and perhaps gain insight into the mechanism of S-nitrosylation. My work in detecting cysteinyl radicals with S K-edge XAS provides a new approach to study the sulfur-based radicals. A future application of significant interest using this technique would be to investigate sulfur-based radical species implicated in a number of enzymatic systems, particularly ribonucleotide reductases (RNR),  an  enzyme  that  catalyzes  the  conversion  of  ribonucleotide  to  deoxyribonucleotide, an essential component for the biosynthesis of DNA. S K-edge XAS can provide a solution to the current difficulties in detecting proposed cysteinyl radicals that initiates the catalytic mechanism and potentially new insights into the proposed mechanism can be gained. Through this thesis, XAS has been successfully used to address the Met oxidation and its implications in disease such as age-related cataracts and Alzheimer’s disease.  S K-edge XAS was also used to investigate the  photochemistry of Met and its oxidized derivatives, MetSO and MetSO2. Photoirradiation of Met under aerobic condition produces oxidized products MetSO and MetSO2, while under anaerobic conditions, Met photoirradiation produces  146  MetS•+.  The mechanism for aerobic photooxidation is believed to involve  photoexcitation of O2 to its singlet excited state. Indirect photooxidation of thioether using photosensitizer to generate thioether radical cation has been proposed as an alternative pathway. My work on the formation of MetS•+ in the absence of O2 and without any sensitizer gives two new pathways. First, a direct one-electron oxidation of Met gives a sulfur-centered radical cation that can react with oxygen to give the oxidized species, MetSO and MetSO2. Second, formation of 3Met* can go through a spin allowed reaction with molecular oxygen to produce MetSO and MetSO2. The photochemistry of MetSO is noteworthy as it reveals the reduction to Met. Photoirradiation of MetSO in presence of O2 produces oxidized product MetSO2 and reduced product Met, and in the absence of O2 only produced Met.  However,  MetSO2 is both oxidatively and reductively inert to photoirradiation.  My  photochemistry work with Met, MetSO and MetSO2 offers significant insights into the mechanisms of photoredox processes that are relevant to the age-related cataract formation.  My photochemistry data presented herein ultimately serves as a  reference to the Met oxidations that occurs in α-crystallins, the predominant component in the cataracts. Thus, it is worthwhile to investigate the photooxidation of α-crystallin proteins using S K-edge XAS. XAS imaging can also be used to investigate the oxidation state of Met in cataract eye lens tissue samples. Another interesting aspect that is worth investigating is the fate of the oxygen in the reduction of MetSO to Met. This can be accomplished with the use of labeled MetSO. Doing a similar in situ XAS photochemistry with the  18  O-  18  O-labeled  147  MetSO and monitoring the decrease of labeled oxygen can provide information on the destination of this cleaved oxygen from the photoirradiation. Also with the development of in situ photochemistry XAS presented in Chapter 4, this new technique gives the potential to investigate the photochemistry of other sulfur compounds that are biologically relevant such as cysteine sulfenic acid, cysteine sulfinic acid, and cysteine sulfonic acid. Probing the fate of these compounds in aerobic and anaerobic conditions may provide some fundamental understanding of the reactivity of these compounds. These in situ investigations are not limited to these compounds, as they can also be used to study S-nitrosothiols. The cleavage of the S-NO bond can provide some useful insights into the mechanism of NO mediated cell signaling. In addition to exploring the photochemistry of Met, I have also explored the metal-induced oxidation of Met, specifically in understanding the Met35 oxidation of amyloid-β (Aβ) peptide.  My work shows that Met35 is oxidized to methionine  sulfoxide in the presence of Cu2+ alone but Fe3+ failed to oxidize Met35.  This  difference in the oxidation behaviour and the lack of evidence for Fe3+ binding to Aβ led me to probe the metal binding site of both Cu2+ and Fe3+. Here I report that the coordination between the two metals is different. While Cu2+ is shown to be in a five-coordinate geometry with a His-rich coordinating environment, Fe3+ is in a sixcoordinate geometry with an oxygen-rich environment. This difference in binding may have some effect to the observed Met35 oxidation differences.  148  In order to gain information regarding Met35 as a potential electron source for the Cu2+ reduction, the Met35 cation radical must be isolated. This requires a set-up in which freezing of the final AβCu2+ mixture is possible, as the Cu2+ solution is mixed with Aβ peptide solution.  Other approaches that can be undertaken in  understanding the Met oxidation in Aβ peptide includes X-ray imaging of amyloid plaques to identify the oxidized form of Met as well as identifying the metals deposited in these plaques. Overall, the work in this thesis has shown that S K-edge XAS is a valuable tool for studying the oxidation of cysteine and methionine. I have shown S K-edge to be a promising tool in investigating sulfur-based radicals. Using S K-edge XAS, I have gained insight into the oxidation of Met and its implication in age-related cataract formation as well amyloid-β peptide oxidation in Alzheimer’s disease.  149  REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9)  (10) (11)  (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27)  Jao, S. C.; Ospina, S. M. E.; Berdis, A. J.; Starke, D. W.; Post, C. 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Karunakaran-Datt, A. and Kennepohl, P., “Redox Photochemistry of Methionine by Sulfur K-edge X-ray Absorption Spectroscopy:  Potential  Implications for Cataract Formation” J. Am. Chem. Soc. 2009, 131, 35773582. (The work presented in Chapter 4 corresponds to this publication)  2. Karunakaran-Datt, A., Martin-Diaconescu, V., and Kennepohl, P.  Chem.  Soc. Rev. In preparation. (Majority of the Chapter 1 will be published as a major component of a broader review article with additional contributions from Vlad Martin-Diaconescu)  3. Karunakaran-Datt, A. and Kennepohl, P., “Redox-active Metal Binding in Amyloid-β and Its Potential Role in Oxidation of Met35” In preparation. (The work presented in Chapter 5 and 6 corresponds to this publication)  4. Karunakaran-Datt, A. and Kennepohl, P. “Protein Sulfur-Centred Radicals: a Sulfur K-edge XAS Detection Method” In preparation. (The work presented in Chapter 3 corresponds to this publication)  167  

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