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Structure-based functional studies of the rhodobacter sphaeroides reaction centre H protein Tehrani, Ali 2003

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Structure-Based Functional Studies of the Rhodobacter sphaeroides Reaction Centre H Protein by Ali Tehrani B . S c , The University of Massachusetts, Amherst, 1994 M.Sc, The University of Massachusetts, Amherst, 1996 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Microbiology and Immunology We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November 2003 © Ali Tehrani, 2003  ABSTRACT  The purple bacterial photosynthetic reaction centre (RC) contains three proteins called R C H , R C M and R C L . The R C H protein comprises three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane ot-helix; a large C-terminal, globular cytoplasmic domain. The roles of these domains in Rhodobacter sphaeroides R C function and assembly was investigated, using a mutagenesis approach that included domain swapping with Blastochloris viridis R C H segments, periplasmic domain deletion and site-directed amino acid changes. Additionally, possible interactions between photosynthetic RC proteins that are thought to protect these membrane proteins from proteolytic digestion in RC complex assembly were evaluated by use of translationally in-frame (non-polar) RC gene-specific deletions. The RC H, RC M and RC L proteins were expressed from plasmids, either alone or in concert with one or both of the others, in a strain of R. sphaeroides that contained chromosomal deletions of all three RC genes. The R C H periplasmic domain was shown to be involved in the accumulation of the R C H protein in the cell membrane, while the transmembrane domain has an additional role in R C complex assembly, perhaps through interactions with R C M . The cytoplasmic domain functions in R C catalytic activity and complex assembly. The R C H cytoplasmic domain surface residues His-126 and His-128 jointly mediate proton transfer into the R C by acting as proton donors at the entrance of the proton transfer pathway. There is a correlation between the amounts of membrane-associated R C H and RC L , whereas R C M is found in the cell membrane independently of R C H and R C L .  Furthermore, substantial amounts of RC M and R C L are found in the soluble fraction of cells only when RC H is present in the membrane. These data were used to propose models of RC catalytic mechanisms, and RC assembly in which the RC M protein accumulates in the cell membrane regardless of the presence of the RC H and RC L proteins, and the RC M protein is a nucleus for addition of RC L followed by RC H in assembly of the RC holocomplex.  iv  TABLE OF CONTENTS Abstract  ii  Table of contents  iv  List of Tables  vii  List of Figures  viii  Abbreviations  x  Acknowledgements  xi  1. INTRODUCTION  1  1.1. Anoxygenic phototrophs 1.2. The anoxygenic purple non-sulfur phototrophic bacterium Rhodobacter sphaeroides 1.3. The photosynthetic apparatus 1.4. Organization and regulation of the R. sphaeroides photosynthesis genes 1.5. Overview of photosynthetic ATP synthesis in anoxygenic purple bacteria 1.6. A closer look at electron an proton transfer pathways in R. sphaeroides R C 1.7. The R C H protein function in photosynthesis and R C complex assembly 1.8. Thesis objectives and approach 2. M A T E R I A L S AND METHODS 2.1. Bacterial strains and growth conditions 2.2. Plasmids utilized 2.2.1. Construction of plasmids used for R C H protein domain swap experiments 2.2.2. Construction of plasmids for site-specific mutagenesis of the R. sphaeroides R C H protein domains 2.2.3. Construction of the plasmids used in R C gene (protein) deletion studies 2.3. Construction of the R. sphaeroides A P U H A A P U C strain  3 1 7 12 14 16 19 19 21  23  27 33 34 36  2.4. Construction of the R. sphaeroides A R C L H strain 2.5. In vitro D N A techniques 2.6. Bacterial conjugation 2.7. Absorption spectroscopy 2.8. Flash spectroscopy 2.9. Kinetics of R C formation 2.10. Electron transfer and proton uptake measurements 2.11. Chromatophore and soluble fraction preparation 2.12. SDS-Gel electrophoresis and western blotting  3. R E S U L T S  43  3.1. Analysis of RC H protein domain swaps on photosynthetic and RC assembly in R. sphaeroides 3.1.1. 3.1.2. 3.1.3. 3.1.4. 3.1.5. 3.1.6. 3.1.7. 3.1.8.  36 37 38 39 39 40 40 40 41  Summary Development of the experimental system Overview of the hybrid RC H proteins Photosynthetic growth of LH2" A P U H A A P U C strains expressing hybrid RC H genes Absorption spectroscopy of LH2" APUHAAPUC strains expressing hybrid RC H genes Photosynthetic growth and flash spectroscopy of A P U H A strains expressing hybrid R C H genes R C H , R C M , R C L proteins levels in membrane and soluble cell fractions Kinetics of RC formation for the APUHAAPUC (pEVtrans) strain  3.2. Site specific mutagenesis and deletion of the R. sphaeroides RC H protein domains 3.2.1. Summary 3.2.2. Overview of the mutant RC proteins 3.2.3. Photosynthetic growth of APUHAAPUC strains expressing RC H periplasmic domain mutations 3.2.4. Absorption spectroscopy of LH2" A P U H A A P U C strains expressing RC H periplasmic domain mutations 3.2.5. Flash spectroscopy of A P U H A strains expressing R C H periplasmic domain mutations 3.2.6. R C H , R C M , R C L proteins levels in membrane and soluble cell fractions of strains expressing RC H periplasmic domain mutations 3.2.7. Photosynthetic growth of APUHAAPUC strains expressing RC H cytoplasmic domain mutations  43 43 43 46 48 50 53 56 59  61 61 61 62 63 66  67 69  vi 3.2.1  Electron transfer and proton uptake measurements for APUHAAPUC strains expressing RC H cytoplasmic domain mutations  3.3 R C gene (protein) deletion studies 3.3.1  Summary  3.3.2  Overview of the R. sphaeroides A R C L H strain and the R C protein deletions Photosynthetic growth of A R C L H strain expressing different combinations of RC genes Absorption spectra of A R C L H strain expressing different combinations of RC genes R C H , R C M , R C L proteins levels in membrane and soluble cell fractions  3.3.3 3.3.4 3.3.5  4. DISCUSSION 4.1. Functional analysis of R. sphaeroides RC H protein domains 4.2. The role of the RC H protein in R. sphaeroides R C complex assembly 4.3. RC-LH1 (core complex) interactions 4.4. Future directions  5. REFERENCES  69 76 76 76 77 79 81  84 84 93 98 98  101  APPENDIX 1 Amino acid alignment of R. sphaeroides and B. viridis R C H proteins  109  APPENDIX 2 Absorption spectra of APUHAAPUC (LH2") intact cells containing plasmid pATSHR or pEVtrans  110  APPENDIX 3 Publications arising from this thesis research  111  vii  LIST OF TABLES  Table 2.1  Bacterial strains  22  Table 2.2  Plasmids used  23  Table 3.1  Summary of RC levels and catalytic activities (for hybrid R C H proteins) measured in steady-state absorption spectra of intact LH2" cells and flash spectroscopy of chromatophores obtained from L H 2 cells  54  Summary of RC levels and catalytic activities (for site-specific periplasmic mutations) measured in steady-state absorption spectra of intact LH2" cells and flash spectroscopy of chromatophores obtained from L H 2 cells.  66  +  Table 3.2  +  viii  LIST OF FIGURES Figure 1.1  Phylogenetic relationships among life forms  1  Figure 1.2  Membrane system o f purple bacterium R. sphaeroides  6  Figure 1.3  Schematic representations of proposed models for the supermolecular organization o f the photosynthetic apparatus o f R. sphaeroides  9  Figure 1.4  Ribbon representation o f the R. sphaeroides R C  11  Figure 1.5  The R. sphaeroides photosynthesis gene cluster  14  Figure 1.6  Schematic illustration o f photosynthetic A T P synthesis in R. sphaeroides  15  Figure 1.7  The catalytic photocycle bf quinone reduction in the R C  18  Figure 2.1  Genetic and restriction map of intermediate plasmid pTZ18U::puhA and pUC19::vpuhA  29  Figure 2.2  Construction of the expression plasmid pATP19P  30  Figure 2.3  Genetic and restriction maps of plasmid pATSHR  31  Figure 3.1  Absorption spectra of APUHAAPUC (LH2") intact cells containing relevant plasmids (expressing hybrid R C H mutants)  45  Amino acid alignment of R. sphaeroides and B. viridis R C H N-terminal sequences, and a representation of hybrid RC H proteins used  47  Photosynthetic growth of APUHAAPUC cells containing relevant plasmids (expressing hybrid RC H mutants)  49  Absorption spectra of APUHAAPUC intact cells containing relevant plasmids (expressing hybrid RC H mutants)  52  Photosynthetic growth of A P U H A cells containing relevant plasmids  55  Figure 3.2  Figure 3.3  Figure 3.4  Figure 3.5  ix Figure 3.6  Western blot involving hybrid RC H mutants probed with R C H , R C M and R C L antisera  58  Figure 3.7  Kinetics of RC formation  60  Figure 3.8  Photosynthetic growth of APUHAAPUC cells containing  Figure 3.9  relevant plasmids (expressing site-specific mutants of RC H periplasmic domain) Absorption spectra of APUHAAPUC intact cells containing relevant plasmids (expressing site-specific mutants of RC H periplasmic domain)  65  Western blot involving site-specific mutations of the R C H periplasmic domain probed with RC H , R C M and R C L antisera  68  Figure 3.10  Figure 3.11  64  Photosynthetic growth and absorption spectra of APUHAAPUC cells containing relevant plasmids (expressing site-specific mutants of RC H cytoplasmic domain)  70  Figure 3.12  Proton uptake and electron transfer  72  Figure 3.13  Chemical representation of the amino acid histidine  73  Figure 3.14  Proton uptake and electron transfer Chemical rescue using imidazole  74 75  Figure 3.15 Figure 3.16  (kAB ) 1  (kAB ) 2  measurements  measurements  Photosynthetic growth of A R C L H cells containing relevant plasmids  78  Absorption spectra of A R C L H cells containing relevant plasmids  80  Figure 3.18  Western blot of A R C L H cells containing relevant plasmids  83  Figure 4.1  Proton path through R C H residues His-126 and His-128  91  Figure 4.2  Chemical structure of TRIS  93  Figure 4.3  Model for R. sphaeroides assembly  Figure 3.17  97  X  ABBREVIATIONS  Ap(Ap )  ampicillin (or ampicillin resistant)  ATP  adenosine 5'-triphosphate  ATPase  adenosine 5'-triphosphatase  bp  base pair  BChl  bacteriochlorophyll  cyt bei  cytochrome be; oxidoreductase complex  HRP  horse radish peroxidase  ICM  intracytoplasmic membrane  kb  kilobases  kDa  kilodaltons  r  Kn(Kn )  kanamycin (or kanamycin resistant)  Ku  Klett unit  LH 1  light-harvesting 1 complex  LH 2  light-harvesting 2 complex  SDS-PAGE  sodium dodecyl sulfate polyacrylamide gel electrophoresis  puc  operon encoding the of L H 2 complex proteins  puf  operon encoding R C M / L and L H 1 complex proteins  puhA  gene encoding the R C H protein  RC  reaction centre  TBS  Tris-buffered saline solution  r  Tc (Tc )  tetracycline (or tetracycline resistant)  QA  RC quinone A  QB  RC quinone B  r  ACKNOWLEDGEMENTS I am indebted to my supervisor, Tom Beatty, for allowing me to mature as a scientist. He is a teacher and a mentor as they should be. Without his support and patience (including "the handshake" after the accomplishment of a task), completing this thesis project and the resulting publications would have been most difficult. I would also like to thank the members of my committee: Drs. Hancock, Fernandez, and Mohn, for their guidance and input into this project. I am grateful to Drs. S. Kaplan, and E. Abresch for providing me with antisera, M . Paddock for purified R. sphaeroides RC, and R.C. Prince for flash spectroscopy experiments. Furthermore, I would like to express my gratitude to M . Paddock, R.C. Prince, and Pia Adelroth for all collaborative experiments. I thank Dr. John Nomellini and Ms. Shelley Small for all the help and support they have provided me over the years. Without a doubt my graduate experience would have not been complete i f it wasn't for my colleagues in the Beatty lab (both past and present). I would like to acknowledge and thank Yves Leduc and Steve Seredick for their friendship and many thought provoking discussions in our times at Wesbrook. Finally, I thank my parents (especially my mother), and Lisa for believing in me and staying patient, as I learn my ways.  This thesis is dedicated to the memory of my grandmother whom forever will be with me.  1  1. INTRODUCTION  Arguably one of the most important biological processes on earth, photosynthesis involves the capture and conversion of light energy into chemical energy (ATP). Within evolutionary schemes there are five distinct bacterial groups (Fig. 1.1), and chloroplasts in eukaryotes that are capable of synthesizing ATP through photosynthesis (40).  Figure 1.1 Phylogenetic relationships among life forms based on large subunit rRNA sequences. Adapted from Woese, C R . et al. (71). Photosynthesis is restricted to the Eucarya (plants and microbes), and Bacteria. Photosynthetic bacteria are found within the cyanobacteria, purple bacteria, green sulfur bacteria, Gram-positive bacteria, and green nonsulfur bacteria.  However, the generation of ATP alone does not fulfill all of the cellular energy requirements, as N A D P H is also needed. Cyanobacteria and chloroplasts use light energy for the transfer of electrons from H2O to N A D P to obtain N A D P H . This +  oxidation of water molecules results in the production and release of molecular oxygen,  2 which categorizes these organisms as oxygenic. Anoxygenic phototrophic organisms produce N A D P H through the oxidation of organic or inorganic compounds. Nevertheless, the general principles of energy transduction are the same for anoxygenic and oxygenic phototrophs (25). In 1941 C B . van Niel described the overall chemistry of photosynthesis with the formulation below, wherein anoxygenic species H2A can for example be H2S, or an organic compound, whereas in oxygenic species H2A is water.  C0  2  + 2 H A + light -» ( C H 0 ) + 2 A 2  2  Phototrophs that oxidize organic compounds to obtain N A D P H typically do not reduce very much C 0 , making this formulation not directly applicable (25, 40). In purple 2  bacterial phototrophic growth, organic compounds can take the place of C 0 and H2A in 2  the above formula. In this case, a revised two-part formula could be written as (the details of light energy conversion to A T P can be found in section 1.5):  light  RC, cyt be 1 > ATP ATPase  organic compound  > N A D P H + oxidized metabolic intermediates > amino acids, nucleotides, etc.  Oxygenic phototrophs contain a variety of light-harvesting antennae that capture and transfer light energy to two types of pigment-protein complexes, both of which are called a reaction centre: 1) PSII designates the reaction centre where water is oxidized; 2) PSI designates the reaction centre where N A D P is reduced (9). Anoxygenic phototrophs +  also contain a variety of light-harvesting systems, but each type of anoxygenic organism  3 1.1 Anoxygenic phototrophs The anoxygenic phototrophic group of bacteria include green sulfur bacteria, green non-sulfur bacteria, heliobacteria, and purple bacteria (Fig. 1.1). Despite many commonalities, these bacteria exhibit a number of differences in the organization of their photosynthetic apparatus, and metabolic characteristics. I provide below a brief summary of three of the four types of anoxygenic phototrophs, followed by a more in depth description of purple phototrophic bacteria, with a focus on Rhodobacter sphaeroides, the subject of this thesis. Green sulfur bacteria. These bacteria are anaerobes that obligately use reduced sulfur compounds as electron donors, while fixing CO2 as a carbon source (27). They primarily capture light though chlorosomes, which are specialized antenna structures found at the cytoplasmic side of the cell membrane. Green sulfur bacteria contain a reaction centre that is thought to be related to the PSI of oxygenic phototrophs (9, 27). Green non-sulfur bacteria. These bacteria are capable of growth under a variety of conditions that include photoautotrophic, photoheterotrophic, and aerobic respiration (58). The fhermophillic bacterium Chloroflexus is the most widely studied member of this group, and has been shown to contain a very similar antenna structure (chlorosomes) to green sulfur bacteria (58). In surprising contrast to the chlorosome similarities, green non-sulfur bacteria contain a reaction centre that is very similar to the purple bacterial reaction centre, and thought to be related to the PSII of oxygenic phototrophs (9). Heliobacteria. The only Gram-positive phototroph, which is also capable of forming spores, these bacteria are strict anaerobes that rapidly die in the presence of oxygen (41). Their reaction centre is thought to be related to the PSI of oxygenic  4 phototrophs (9). Given that they are the most recently discovered photosynthetic organism, not much is yet known about their mechanism of carbon assimilation or metabolic characteristics, other than they require an organic carbon source for growth (9). Purple bacteria. As the metabolically most versatile and easily cultivated of the anoxygenic phototrophs, these bacteria have been the subject of extensive structural and genomic studies (9). Purple phototrophic bacteria contain a reaction centre that is thought to be related to the oxygenic PSII reaction centre, and very similar to the reaction centre of green non-sulfur bacteria (20). On the basis of the ability to oxidize reduced sulfur compounds for CO2 fixation, purple bacteria are subdivided into sulfur and nonsulfur groups. Under highly aerobic conditions, when growth occurs by chemotrophic respiration, the cell membrane of most purple bacteria is morphologically similar to other Gram-negative species such as Escherichia coli (20, 40). However, a lowering of the oxygen partial pressure (typically to <1 kPa), results in the formation of invaginations in the cytoplasmic membrane, which is where the photosynthetic apparatus is found (Fig. 1.2) (20). This elaborate intracytoplasmic membrane system (ICM) is continuous with the plasma membrane and (depending on the species) has been shown to consist of either finger-like intrusions, vesicles, tubular structures, or relatively flat sheets known as lamellae (20). Studies on the purple non-sulfur bacterium Rhodobacter sphaeroides have provided a wealth of information on the fundamentals of subjects such as membrane biogenesis, electron and proton transfer reactions in the membrane, and properties of membrane proteins. The ability of this bacterium to grow under a variety of conditions (i.e. chemotrophically or phototrophically), such that mutations that abolish  5 photosynthetic growth are not lethal, makes this organism ideal for the study of bacterial photosynthesis (20, 36, 48, 51, 66, 74).  6  ICM  Figure 1.2 Membrane system of purple bacterium R. sphaeroides. Left panel, electron micrograph of R. sphaeroides, depicting intracytoplasmic membrane invaginations (ICM). Right panel, schematic representation of the photosynthetic membrane of R. sphaeroides, with a closer look at an ICM (see Fig 1.6 for components of the ICM). Figures adapted from www.eawag.ch/research e/ and www.nottingham.ac.uk/~pdzres respectively.  7  1.2 The anoxygenic purple non-sulfur phototrophic bacterium Rhodobacter  sphaeroides On the basis of 16S rRNA sequence, R. sphaeroides belongs to the a-subgroup of Proteobacteria, and forms almost spherical rod-shaped cells, which frequently occur in pairs or chains. Under anaerobic conditions, its characteristic yellowrbrown pigmentation is due to the photosynthetic pigments bacteriochlorophyll a, and the carotenoid spheroidene (27). Anaerobically and with light, R. sphaeroides grows phototrophically, although it can also carry out anaerobic respiration in the dark with the addition of a terminal electron acceptor such as dimethyl sulfoxide. Aerobically in the dark, R. sphaeroides utilizes a number of organic compounds such as malate as a carbon source for respiration (74).  1.3 The photosynthetic apparatus Embedded within the I C M , pigment protein complexes of the R. sphaeroides photosynthetic apparatus comprise the light-harvesting antennae called LH2 and LH1, and the reaction centre (RC) complex, which operates as a light-driven two-electron/twoproton quinone reductase (20, 51). X-ray crystallography of LH2 complexes revealed symmetrical pigment-protein ring structures (34, 43), which transfer light energy to the LH1 complex (66). The LH1 complex transfers light energy to the RC, and a variety of experiments indicate the existence of a "core" LH1/RC supercomplex (39). Currently, the exact organization of this supercomplex is the subject of extensive research and debate. Recently, Vermeglio and colleagues reported that in R. sphaeroides, LH1 partially encircles the RC, forming a crescent structure that may allow movement of  8 quinones between the RC and cytochrome bc\ (Fig. 1.3A) (69). Contrary to this model, other research suggested that LH1 forms a closed circular structure around the RC, with LH2 complexes surrounding the monomeric RC/LH1 supercomplex (Fig. 1.3B) (14).  Figure 1.3 Schematic representations o f proposed models for the supermolecular organization o f the photosynthetic apparatus o f R. sphaeroides. A M o d e l depicting a pair o f R C s , each surrounded by crescent L H 1, associating with cytochrome bc . The core is surrounded by peripheral antenna complexes ( L H 2). A l s o shown is the P u f X protein (solid dot) located between R C and Cyto beh B M o d e l depicting an R C surrounded by a closedcircle L H 1 complex. Although not shown here, in this model the P u f X protein forms a gate in L H 1 for quinone exchange in the R C ((38). Figure adapted from H u et al. (73) t  10 Light harvesting 2 complex. Classified as an accessory antenna complex, variable amounts of LH2 can be found in the membrane depending on growth conditions. Many species of purple bacteria do not contain a LH2 complex, and mutants of R. sphaeroides that lack LH2 are capable of photosynthetic growth (15). The basic structural unit of this complex consists of a heterodimer of two integral membrane polypeptides known as the a and the P subunits (encoded by pucA and pucB genes), which non-covalently bind three bacteriochlorophyll (BChl) a and one carotenoid molecule (14, 43). Following oligomerization, the subunits form a ring-like structure in which BChl a molecules are arranged as a ring of interactive dimers that absorb light at 850 nm, and monomers that absorb light at the 800 nm wavelength. X-ray diffraction studies have revealed that depending on the species, LH2 rings consist of oligomers of eight or nine a/p subunits (34, 43). A n (a/p)g ring has been demonstrated in a projection structure determined from 2-D crystals of the R. sphaeroides LH2 complex (see J. Mol. Biol. 282:833-845, 1998). Light harvesting 1 complex. Although similar to LH2, the LH1 core antenna complex is clearly distinct as it absorbs light at a different wavelength, aggregates into a larger complex, and LH1 is found in a fixed stoichiometric ratio to the RC. Structurally, this complex is made up of approximately 16 pairs of a/p polypeptide subunits, with each pair binding two BChl a molecules and two carotenoid pigments (20). The pufA and pufB genes encode the L H l a and P protein subunits (26). Unlike LH2, the oligomerized BChl a dimers in LH1 absorb light at the 870 nm wavelength, and transfer the resultant excitation energy directly to the RC. It is not clear how the different oligomerization and/or protein environments of the LH1 and LH2 BChl dimers result in their different wavelength absorption properties. In 2000, Hunter and colleagues reported yet another  11  major difference between L H 1 and L H 2 , in that they found the P subunit in L H 1 to be more bent, which would direct its N-terminus toward the enclosed R C (16).  Reaction centre.  A widely studied integral membrane pigment-protein complex,  the R C o f R. sphaeroides is composed o f three protein subunits named R C H , R C M and R C L , and nine cofactors (Fig. 1.4) (23). Certain purple bacterial species such as Blastochloris  viridis, contain a cytochrome c subunit as the fourth protein component o f  theRC(18).  F i g u r e 1.4 Ribbon representation of the R. sphaeroides R C . The R C complex is comprised of three proteins subunits (RC H, R C M , and RC L ) , and nine cofactor molecules. Figure adapted from: http://physics.ucsd.edu/~raifeher/rc_ribn.html  12 High-resolution crystal structures of the RCs of B. viridis and R. sphaeroides revealed that RC M and RC L each have five a-helical hydrophobic transmembrane segments, while R C H contains only one such transmembrane segment (36). The R C H protein does not interact directly with cofactors, and can be conceptually divided into three domains: 1) an ~11 amino acid N-terminal domain located on the periplasmic side of the inner membrane of these Gram-negative cells; 2) a single transmembrane a-helix; 3) a C-terminal, large, globular cytoplasmic domain (36). The R C M and RC L proteins, which are homologous and have a pseudo-two-fold axis of symmetry, provide a scaffolding within which the RC associated cofactors are arranged in two symmetrical branches commonly referred to as the A and the B branches. These cofactors are: two BChl a which form a strongly interacting dimer known as the "special pair" (sometimes called D), two accessory BChl a which are in close proximity 2_|_  to the special pair, two bacteripheophytins, one non-heme iron atom (Fe ), and a pair of quinones ( Q , Q B ) (4, 11). A  1.4 Organization and regulation of the R. sphaeroides photosynthesis genes. In R. sphaeroides and other purple bacteria, the genetic information required for pigment biosynthesis and the production of photosynthetic pigment-binding proteins is contained within a cluster of genes, commonly referred to as the photosynthesis gene cluster (40,46). The arrangement of the R. sphaeroides photosynthesis gene cluster is shown in Figure 1.5. The a and B polypeptides of the LH1 complex, along with the R C L and RC M protein subunits, are encoded within thepuf operon (26). Relative to thepuf operon, the puhA gene, which encodes the R C H protein, is located at the opposite end of  the cluster. The LH2 complex is not essential for photosynthesis and the genes encoding the a and (3 polypeptides of this complex are found within the puc operon (40, 46). This operon is not part of the photosynthesis gene cluster, and is located ~20 kb 3 of the puhA gene. The expression of photosynthesis genes in R. sphaeroides is controlled through the function of several transcriptional regulatory circuits, which are affected by changes in cellular oxygen tension, as well as changes in light intensity (7, 49). Generally, under high oxygen conditions the expression of the photosynthetic apparatus is repressed due to the binding of a repressor protein (PpsR) to a conserved palindrome sequence on the chromosome (48, 75). Additionally, the expression of the R C and L H structural proteins is regulated by a two-component signal transduction system (RegBA), which up-regulates the expression of the puf, puc, and puhA genes in response to a drop in oxygen partial pressure (22, 47, 62). Although not fully understood, the expression of the photosynthesis genes is also affected by light intensity through the AppA protein. Recently Bauer et al. proposed a model in which AppA acts as a transcription factor that controls light repression of photosynthesis gene expression, by mediating the binding of D N A by PpsR. That is, under low O2 and low (blue) light conditions, AppA inhibits D N A binding by PpsR, hence allowing the expression of photosynthesis genes. Under low light conditions, the transcription of puc, puf, and the puhA genes is enhanced, while high light has been reported to repress puc operon gene transcription (42).  14 The above brief summary neglects many details of the fervid and sometimes controversial field of purple bacterial photosynthesis gene expression. The interested reader is directed to recent reviews (6, 29).  ( rn  \ <ml i in i f I m i n i I J 11) i II ti puf  puhA  • 111)11  II  operon  Figure 1.5 The R. sphaeroides photosynthesis gene cluster. The arrows within the genes indicate the direction of transcription of various genes, and the arrows above the map indicate the genes grouped into operons. Figure reproduced from Naylor et al (46)  1.5 Overview of photosynthetic ATP synthesis in anoxygenic purple bacteria Generally, purple bacterial photosynthesis involves the capture of light energy by the light harvesting antennae system and transfer to the RC, which is the site of primary charge separation and initiation of proton transfer across the cytoplasmic membrane in photosynthetic energy transduction (51). These processes are best understood in R. sphaeroides,  which I have used as a model organism for my research.  As shown in Figure 1.6, electron transfer reactions in the RC are coupled to proton uptake from the cytoplasm, to convert a quinone (QB) to a quinol  (QBH.2).  The  release of this quinol from the RC and into a quinone pool is facilitated by the PufX  15 protein, which is thought to provide a way by which quinones can enter and leave the R C (38). The oxidation of Q B H 2 by the cytochrome bci oxidoreductase reduces cytochrome  C2 and results in the release of protons into the periplasm. The electrons from reduced cytochrome C2 are transferred to the R C . Thus, electron flow in purple anoxygenic photosynthesis is cyclical, while the coupled translocation of protons across the cytoplasmic membrane generates a proton-motive force that drives the generation of ATP by the ATP synthase (51).  Periplasm  LH2  LH1/RC  cyt c  2  Cyt  b/  Cl  ATPase  Figure 1.6 Schematic illustration of photosynthetic ATP synthesis in R. sphaeroides. The various complexes are shown as imbedded in the ICM lipid bilayer, with their names given in boxes. See text for details. Adapted from Fejes etal (24).  1.6 A closer look at electron and proton transfer pathways in the R. sphaeroides R C As noted above, anoxygenic photosynthesis involves the reduction and protonation of the Q B quinone in the R C . The double reduction of this quinone requires two sequential light-induced electron transfer reactions, in concert with the translocation of two protons from the cytoplasm into the R C (Fig. 1.7A) (51). Electron transfer reactions commence with the transfer of an excited electron from the special pair to a bacteriopheophytin. The reduced bacteriopheophytin donates an electron to the nearby Q A quinone, which in turn passes it to QB, to form a relatively stable semiquinone radical. The R C in the membrane is oriented such that the special pair is close to the periplasm, and the now positively charged special pair is neutralized by the transfer of an electron from cytochrome C2, which is mobile in the periplasm. Upon the absorption of a second photon, another electron is transferred to QB, and two protons are obtained from the cytoplasm (see below for details), yielding the Q B H quinol (51). 2  Proton pathways, consisting of protonatable amino acid residues, from the cytoplasmic surface to the Q B head group, have been the subject of extensive investigations. High resolution X-ray crystal structure data for the R. sphaeroides R C , revealed three possible proton pathways, each with distinct entry points for protons (Fig. 1.7B)(1,65). The Q B molecule is embedded within the R C L protein with several polar and acidic residues in its immediate surrounding. Site-directed mutagenesis studies revealed that R C L residues Glu-212, Asp-213, and Ser -223 are necessary for the transfer of protons to Q B (Fig. 1.7B). Specifically, the first electron transfer from Q  A  to Q (step 2, B  Fig. 1.7A) is coupled to the protonation of Glu-212 (54, 55, 67). The first protonation of  17 Q B however occurs during the second electron transfer reaction (step 4, Fig 1.7A) as QB" obtains a proton from Ser-223, yielding the intermediate (QBH)". The proton accepted by Glu-212 during the first electron transfer reaction is then donated to the (QBH)", forming QBH  2  (51).  Recently, studies by Okamura and colleagues suggested that proton transfer to Q B proceeds through a single pathway proximal to the RC H protein surface residues His126, His-128, and Asp-124 (Fig. 1.7A). This conclusion was derived from observed inhibitory effects on proton transfer upon binding of Cd or Z n +  2 +  to these residues (5, 53).  However, the reason for the inhibitory affect of metal binding to R C H residues Asp-124, His-126, and His-128 was unclear. That is, it was not clear whether one or more of these three R C H surface residues is a proton carrier, or if the presence of the bound (positively charged) metal ion has an electrostatic effect that changes the p K of other protein side A  chains that act as proton carriers in the pathway from the cytoplasm to Q B (5, 53).  18  cyt*  0,  _cyi 3*  D Q A QB  DQAQB  OQA  i  AB .ho  D QA (QBH2) IA(QE  DQAQB  DUA(QBH)  DQAQB  CY  *  B t  His  O 2+  Asp M17  _  _  . Glu HI 73  ~ ^ A s p V^ABp  P3 *'  f"\  L210  \H126  \  '  V-S  Asp  H124  • H170 •  • *•  •  VPI  1  His H128 l ^ H l s kH68  * J Qlu  *  Asp M240  —^H224  Figure 1.7 A The catalytic photocycle of quinone reduction in the R C . The QB quinone is reduced in two sequential electron transfer reactions, defined by rate constants k B , and k , and is protonated by H ( l ) , and H (2). The reduced Q B H leaves the R C and is replaced by an exogenous quinone. Electrons lost by D (the special pair) are replaced by cyt c B Schematic illustration of the Q site depicting three possible proton transfer pathways (labelled PI-3). Figure Adapted from Okamura et al. (51) 2  A  +  AB  +  2  2  B  19  1.7 The RC H protein function in photosynthesis and RC complex assembly As noted above, X-ray crystallography data for the B. viridis and R. sphaeroides RCs suggest that the R C H protein subunit can be conceptually divided into a periplasmic domain, a transmembrane domain and a cytoplasmic domain. Removal of the R C H protein from the R. sphaeroides R C in vitro was shown to destabilize the Q B quinone, although electron transfer from the special pair to Q A was possible (17). Gross disruptions of the R C H gene renders the R C undetectable in absorption spectra and abolishes photosynthetic growth in several species, although traces of RC primary charge separation were reported in R. sphaeroides and Rhodospirillum rubrum mutants. Interestingly, these RC H gene disruptions reduced the levels of the LH1 complex (12, 13, 64, 72).' It was suggested that the RC H protein is required in vivo to provide chaperone-like activities for membrane insertion of the RC M and R C L proteins to form the R C holocomplex (64, 68), and that this holocomplex is required for maximal levels of LH1 (3, 13, 72). However, the details of how the R C H protein might participate in these proposed activities, and which segment(s) of the protein are involved, were unknown.  1.8 Thesis objectives and approach The main focus of my research was to investigate and elucidate the function of the R. sphaeroides RC H protein and its domains, relative to the assembly of the R C complex and photosynthetic catalytic activity. The results of these experiments led me to also study the possibilities of interactions between the R C H , R C M , and RC L proteins that protect them from degradation during assembly of the RC holocomplex. This thesis reports my findings in the following areas: 1) Development of host strains and plasmids  20  for expression of various combinations of heterologous, homologous, and mutant RC and LH1 genes; 2) Functional evaluation of the structurally similar R C H protein from Blastochloris viridis when expressed in R. sphaeroides; 3) Analysis of RC H domain swaps between R. sphaeroides and B. viridis on photosynthesis and R C assembly; 4) R C H site-specific mutagenesis; 5) Studies of strains that expressed RC H , R C M and RC L genes singly, or in pairs. Collectively, these results allowed me to propose a general role in assembly and catalytic function for each R C H protein domain, and formulate a model for the assembly of the RC holocomplex in R. sphaeroides.  21  2. MATERIALS & METHODS  2.1 Bacterial strains and growth conditions Table 2.1 lists the E. coli and R. sphaeroides strains utilized in this thesis. Subclonings were preformed in E. coli strains DH5ct, RB404 or DH10B. The E. coli strain SI7-1 was used as a mobilizing strain for conjugation with R. sphaeroides. Additionally, E. coli strain DH10B and the E. coli helper strain HB101(pRK2013) were used for transfer of plasmids to R. sphaeroides in tri-parental conjugations (see section 2.6 for details on bacterial conjugation) (19). The R. sphaeroides APUHAAPUC and A R C L H mutant strains were constructed by step-wise introduction of chromosomal deletions into the puc operon (to obtain APUHAAPUC), and puf I puc operons (to obtain ARCLH), of the R. sphaeroides A P U H A strain (see sections 2.3 and 2.4 for details). The R. sphaeroides A P U H A strains contains an in-frame chromosomal deletion of the puhA gene (12). E. coli was grown aerobically in Luria-Bertani medium (LB) (60), at 37 °C. R C V medium (8) supplemented with 1 pg ml" nicotinic acid was used for the growth of R. 1  sphaeroides at 30 °C. For semi-aerobic growth, cultures were grown in Erlenmeyer flasks filled to 80% of the nominal volume and shaken at 150 rpm. Photosynthetic cultures were inoculated with cells grown under semi-aerobic conditions and incubated in completely filled screw-cap tubes, illuminated with -275 uE m" s" of light from halogen 2  1  lamps (Capsylite; Sylvania). B. viridis was grown photosynthetically in YPS medium (70).  22 For plasmid selection, media were supplemented with antibiotics at the following concentrations: ml" ; 1  R. sphaeroides,  tetracycline-HCl at 2 jxg ml" , kanamycin-sulfate at 25 pg 1  E. coli, tetracycline-HCl at  10 pg ml" , kanamycin-sulfate at 50 pg ml" , ampicillin 1  1  at 150 pg ml" . 1  Turbidities of liquid cultures were measured with a Klett-Summerson colorimeter equipped with a red (#66) filter (1 Klett unit = ~ 10 cells ml" ). 7  1  Table 2.1 Bacterial strains  Strains  E.  Relevant characteristics  coli  Source or reference  -  DH5a  F", A(lacZYA-argF)  DH10B  Invitrogen  HB101(pRK2013)  F~, mcrA, recAl, endAX, araA 139, A/acX74 pRK2013 contains RK2 transfer genes  RB404 S17-1  dam' pro, res',  ((10)  2.4.1  Wild type strain  (12)  APUHA  RC",LH1 ,LH2  (12)  APUHAAPUC  RC", L H 1 , LH2", K a n  ARCLH  RC", LH1", LH2"  R.  deoR,  mob  +  recAl,  endAl  Invitrogen (19) (63)  sphaeroides +  +  r  This study This study  23 2.2 Plasmids utilized Table 2.2 lists the plasmids utilized in this thesis. A detailed explanation for the construction of the relevant plasmids follows.  Table 2.2 Plasmids used Strains or Plasmids pAli::puhA  Relevant characteristics pTZ::puhA with Hind. I l l to Xba I of the multiple  Source or reference This study  cloning site removed pAH2  pUC19 with Kpn I to Hinc II of the multiple  This study  cloning site removed, and Hind III changed to EcoR I pAH2::705BA  pAli2 containing a 3.2-kb Pst I D N A fragment  (37)  encoding R. sphaeroides puc operon sequences with 705 bp of the pucBA genes replaced with.the neo gene pAH2::puf  pAH2 containing a 4.57-kb EcoR I fragment,  This study  encoding the R. sphaeroides pufQBALMX genes pAH2::PUFDEL  pAH2 containing a puf operon deletion extending  This study  from 13 bp 5' of the pufB start codon to 19 bp 3' of the pufXstop codon, as a 2.1-kb Pst I D N A fragment pAH2::DELPUC  pAH2::705BA with the neo gene removed through  This study  digestion with Kpn I and self-ligation pATP19P  pRK415 containing R. sphaeroides puc promoter as (31) a 0.75-kb Hind III fragment  pATSHR  pATP19P containing BamR I fragment of  This study  pTZ18U::puhA pATVHR  pATP19P containing R. viridis R C H gene as a 1.5- This study kb EcoRI fragment  24 pDG4B  Contains B. viridis RC H gene as 3.5-kb BamHl  J. Farchaus  fragment pAPeri  pTemp2 with codons G-3 though F-10 removed  pEAPeri  pATP19P containing the BamR I fragment of  This study This study  pAPeri pEH126A  pATP19P containing the BamR I fragment of  This study  pH126A pEH128A  pATP19P containing the BamR I fragment of  This study  pH128A pEPUF  pATP19P containing the EcoR I fragment of  This study  pAli2::puf pESHPUF  pATSHR containing the EcoR I fragment of  This study  pAli2::puf pESHPUFL"M"  pATP19P containing the EcoR I fragment of  This study  pAli2::PUFL"M" pESHPUFL"  pATP 19P containing the EcoR I fragment of  This study  pAli2::PUFL" pESHPUFM"  pATP19P containing the EcoR I fragment of  This study  pAli2::PUFM" pEStiplMA  pATP19P containing the BamR I fragment of  This study  pStiplMA pEStiplMT  pATP19P containing the BamR I fragment of  This study  pStiplMT pEStip2M  pATP19P containing the BamR I fragment of  This study  pStip2M pEVtrans  pATP19P containing the BamR I fragment of  This study  pVtrans pEVtip  pATP19P containing the BamR I fragment of pVtip  pH126A  p T Z l 8U::puhA with H-126 replaced with A codon  This study  pH128A  pTZ18U::puhA with H-128 replaced with A codon  This study  This study  25 pRK415  Broad host-range cloning vector, Tc  (30)  pNGHl  Suicide vector, ColEi-onT, RP4-onT, sacB, K m  r  (50)  p N G H l containing Pst I fragment of pNGHl::DELPUC  This study  pAli2::DELPUC p N G H l containing Pst I fragment of  pNGHl::PUFDEL  This study  pAli2::PUFDEL pAli2 containing a 3.45-kb Pst Ipuf operon D N A  pAH2::PUFL"M"  fragment with translationally in-frame deletions in  This study  the pufM and pufL genes pAli2::PUFL"  pAli2 containing a 4.2-kb Pst I puf operon D N A  This study  fragment with a translationally in-frame deletion in the puf L gene  pAli2::PUFM"  pAli2 containing a 3.8-kb Pst I puf operon D N A  This study  fragment with a translationally in-frame deletion in the puf M gene Suicide vector, pBR325 derivative, Mob , A p , +  pSUP203 pSUP203::705BA  Cm , Tc r  r  (63)  r  pSUP203 containing Pst I fragment of  This study  pAH2::705BA pStiplMA  pTZ18U::puhA with N-9 replaced with L codon  This study  pStiplMT  pTZ18U::puhA with T-5 replaced with V codon  This study  pStip2M  pTZ18U::puhA with both T-5 and N-9 replaced by  This study  V and L codons, respectively pTemp2  pAli::puhA with two Hinc II sites engineered at G-  This study  3 &F-10/D-11 codons pTZ18U pTZ18U::mspuhA  pTZ18U::puhA  Cloning vector, Ap  Pharmacia  pTZ18U::puhA with an engineered Dde I site at T-  This study  33 codon pTZ18U containing the R. sphaeroides R C H gene (12) as a 1.34-kb BamR I fragment  26 pTZ18U::puhA2.1  pTZ18U::puhA with an engineered EcoR V site at  This study  L-12 codon pTZ18U::xbsph  pTZ18U::puhA with an engineered Xba I site at F-  This study  10 pUC19  Cloning vector, A p  r  (44)  pUC19::vpuhA with an engineered Dde I site at RpUC19::mvpuhA  34 codon  This study  pUC19 containing the B. viridis R C H gene as a pUC19::vpuhA  1.5-kb EcoR I fragment  This study  pUC19::vpuhA with an engineered Xba I site at L pUC19::xbvir  10  This study  pUC19 encoding R C H gene with the B. viridis pVCSP  cytoplasmic residues (starting at Glu 35) in place of  This study  the R. sphaeroides sequence (after Glu 34) as a 1.4kb BamR I fragment pVPSCA  pUC19 containing a R C H gene encoding the B.  This study  viridis periplasmic and transmembrane domains (has P as 3 4 ^ codon) and the R. sphaeroides cytoplasmic domain pVPSCB  p V P S C A with P-34 changed to R (wild type)  This study  pVPSCB2  pVPSCB with an engineered EcoR V site at D - l 1  This study  codon pVtip  pUC19 containing a hybrid R C H gene encoding  This study  the B. viridis periplasmic amino acids in place of the corresponding R. sphaeroides sequence as a 0.9-kb BamR I fragment pVtrans  pTZ18U containing a hybrid puhA gene encoding the B. viridis transmembrane domain flanked by the R. sphaeroides periplasmic and cytoplasmic domains as a 1.2 kb-BamR I fragment  This study  27  2.2.1 Construction of plasmids used for RC H protein domain swap experiments Site-directed sequence changes were introduced by use of the QuikChange kit (Stratagene), and all mutations were D N A sequenced. Several intermediate plasmids were used to create the mutant RC H expression vectors as described below. The proteins produced by each of the mutants are represented in Fig. 3.2B. The R. sphaeroides R C H gene in plasmid pTZ18U::puhA (Fig. 2.1A) was subcloned as a 1.3-kb BamR I fragment into pATP19P (Fig. 2.2), yielding plasmid pATSHR (Fig. 2.3). The B. viridis RC H gene of plasmid pDG4B was transferred to pTZ18U as a BamR I to Xma III fragment, subcloned into pUC19 as a 1.5-kb EcoR I fragment, yielding plasmid pUC19::vpuhA (Fig. 2. IB), and transferred from pUC19::vpuhA as an EcoRI fragment to pATP19P to yield p A T V H R . The plasmid pEVtip encodes the B. viridis periplasmic amino acids Met-1 to Asp11 in place of the corresponding R. sphaeroides sequence (see shaded residues in Fig. 3.2A)'. To construct the plasmid pEVtip, Xba I sites were engineered at the RC H Leu-10 codon (TTA  CTA) of plasmid pUC19::vpuhA, and Asn-9 ( A A C  A A T ) and Phe-10  (TTC -> CTA) codons of plasmid pTZ18U::puhA, to yield the intermediate plasmids pUC19::xbvir and pTZ18U::xbsph, respectively (both plasmids also contain Xba I sites 5' of their respective RC H genes). After an Xba I digest the 845-bp D N A fragment from plasmid pTZ18U::xbsph was ligated to a 2.7-kb fragment from the plasmid pUC19::xbvir, resulting in plasmid pVtip. The hybrid RC H gene in plasmid pVtip was  28 subcloned as a 900-bp BamR I fragment into the expression vector pATP19P to yield pEVtip.  29  EcoRI  Figure 2.1 Genetic and restriction map of intermediate plasmid. A Plasmid pTZ18U::puhA. The R. sphaeroides puhA gene is represented as a filled labelled arrow. This puhA gene was subcloned as a 1.34-kb BamR I DNA fragment, with the start codon (ATG) being at the 474 base pair. This plasmid is ampicillin resistant (Ap ). B Plasmid pUC19::vpuhA. The B. viridispuhA gene is represented as filled labelled arrow. This puhA gene was subcloned as a ~1.5-kb EcoR I DNA fragment, with the stop codon being at the 820 base pair. This plasmid is ampicillin resistant (Ap ). th  1  th  r  30  ^  i  r —I—i—1  '  - 1  Hind III p  puc operon promoter  1  I Pst I | xbal I Hinc UJSal I Sma I  Hind III  A ~ 0.7-kb Hind III fragment containing the R. sphaeroides puc operon promoter was sub-cloned from plasmid pUI612 (37), into plasmid pRK415, yielding plasmid pATP19P  rMCS  Sma I  Figure 2.2 Construction of the expression plasmid pATP19P. Relevant restrictions sites are provided. The bent arrows indicate the location of the R. sphaeroides puc operon promoter, which was used to transcribe all hybrid and mutant puhA genes in this thesis (37). Filled arrow in pATP19P represents tetracycline resistance.  31  Smal Stul  Figure 2.3 Genetic and restriction maps of plasmid pATSHR. See sections 2.2.1 and 2.2.3 for construction and use. Bent arrows indicate the puc operon promoter, which drives the transcription of the R. sphaeroides puhA gene.  32 Plasmid pEVtrans encodes the R. sphaeroides periplasmic domain from Met-1 to Asp-11, followed by the B. viridis transmembrane domain from Ile-12 to Arg-34 (Fig. 3.2A), and the R. sphaeroides cytoplasmic domain from Glu-34 onward. This plasmid was created by initially introducing Dde I sites at the location corresponding to the junction between the transmembrane and cytoplasmic domains of both the R C H genes in plasmids pUC19::vpuhA and pTZ18U::puhA. This was done by changing the R. sphaeroides Thr-33 codon (ACC -> ACT) yielding plasmid pTZ18U::mspuhA, and the B. viridis Arg-34 codon (CGT  CCT) to yield plasmid pUC19::mvpuhA. A 3.6-kb  Hind III (filled in with T4 D N A polymerase) to Dde I fragment containing the D N A sequence of the R. sphaeroides cytoplasmic domain was obtained from pTZ18U::mspuhA, and ligated to a 240-bp Pvu II to Dde I fragment (encoding the B. viridis periplasmic and transmembrane domains) from pUC19::mvpuhA to yield plasmid pVPSCA. The D N A sequence of plasmid pVPSCA R C H codon 34 was changed from C C T back to CGT (restoring Arg-34), creating pVPSCB. EcoR V sites were introduced at the B. viridis R C H Asn-11 codon (GAC -> GAT) in pVPSCB, to yield pVPSCB2, and at the R. sphaeroides R C H Leu-12 codon (CTG  ATC) in pTZ18U::puhA, yielding  p T Z l 8U: :puhA2.1. The 800-bp EcoR V to Hind III fragment of pVPSCB2 was ligated to the 3.2-kb fragment of plasmid pTZ18U::puhA2.1, yielding pVtrans. A 1.4-kb BamH I fragment was subcloned from pVtrans into pATP19P to yield pEVtrans. To obtain plasmid pEVPSCB, which contains a R C H gene encoding the B. viridis periplasmic and transmembrane domains (Met-1 to Arg-34; see Fig. 3.2A) followed by R. sphaeroides sequences from Glu-34 onward, the 1.4-kb EcoR I fragment from plasmid pVPSCB was sub-cloned into the expression vector pATP19P.  Plasmid pEVCSP encodes the R. sphaeroides R C H periplasmic and transmembrane domains (amino acids Met-1 to Glu-34; see Fig. 3.2A) followed by the B. viridis R C H cytoplasmic domain (from Asp-36 onward). Plasmid pEVCSP was constructed by ligation of a 3.4-kb Kpn I (resected with T4 D N A polymerase) to Dde I fragment from pTZ18U::mspuhA to a 820-bp Pvu II to Dde I D N A fragment from pUC19::mvpuhA, to yield plasmid pVCSP. The 1.4-kb BamR I fragment from plasmid pVCSP was subcloned into pATP19P to yield pEVCSP.  2.2.2  Construction of plasmids for site-directed mutagenesis of the R.  sphaeroides RC H protein domains Site-directed sequence changes were introduced by use of the QuikChange kit (Stratagene), and all mutations were D N A sequenced. Plasmid pEAPeri encodes a mutant R. sphaeroides R C H protein in which Val-2 is followed by Asp-11 (codons 3 through 10 of the R C H periplasmic domain were removed; see Fig. 3.2A). Plasmid pEAPeri was made by first introducing two Hinc II restriction sites, one at the Gly-3 codon (GGT -> A A C ) and the other at the Phe-10/Asp11 codons (TTC/GAT to GTC/GAC) in plasmid pTZ18U::puhA, resulting in plasmid pTemp2. After Hinc II digestion, the D N A segment encoding Gly-3 to Phe-10 was removed and the plasmid re-ligated to yield plasmid pAPeri. A 1.3-kb BamR I fragment from pAPeri encoding the mutant R C H gene was subcloned into the expression vector pATP19P (see section 2.2.1 for details on this plasmid) to obtain pEAPeri. Mutant R C H genes in plasmids pStiplMT (Thr-5 [ACT] -> Val [GTT]) and pStiplMA (Asn-9 [AAC] -> Leu [CTC]) were generated in pTZ18U::puhA (see Fig  34 3.2A). Plasmid pStip2M was similarly generated by simultaneous replacement of Thr-5 and Asn-9 with Val and Leu codons, respectively (Fig. 3.2A). These mutant RC H genes were transferred to pATP19P as BamR I fragments. Mutant R C H genes in plasmids pH126A (His-126 [CAC] -» Ala [GCG]) and pH128A (His-128 [CAC] -» Ala [GCG]) were generated in pTZ18U::puhA. These mutant RC H genes were transferred to pATP19P as BamR I fragments yielding plasmids pEH126A and pEH 128A respectively.  2.2.3 Construction of the plasmids used in RC gene (protein) deletion studies Site-directed sequence changes were introduced by use of the QuikChange kit (Stratagene), and all mutations were D N A sequenced. Plasmids pEPUF and pESHPUF were constructed by sub-cloning of a 4.57-kb EcoR I fragment, encoding the R. sphaeroidespufQBALMX genes (Fig. 2.4A) into plasmids pATP19P and pATSHR (see Materials and Methods section 2.2.1 for details on these plasmids) respectively. Thus pEPUF contains only the puf operon, whereas pESHPUF contains the puf operon and the puhA (RC H) gene. Plasmid pESHPUFL" was constructed by insertion of a 4.2-kb EcoR I D N A fragment (Fig. 2.4B), which contains a translationally in-frame deletion of the RC L (puf L) gene that removed 353 bp between Kpn I (resected with T4 D N A polymerase) and BsaB I sites, into pATSHR. This plasmid also contains the puhA gene. Plasmid pESHPUFM" was constructed by insertion of a 3.8-kb EcoR I fragment (Fig. 2.4C), which contains a translationally in-frame deletion of the R C M ipufM) gene that removed 767 bp between Rsr II and BstXl sites that were joined by ligation with the  35 linkers 5 ' - G A C C G G C C G A C G A T A T C A T C T G G - 3 ' and 3 ' - G C C G G C T G C T A T A G T A 5', into pATSHR. This plasmid also contains the R C H (puhA) gene. Plasmid p E S H P U F L M " was constructed by insertion of a 3.45-kb EcoR I fragment, which contains the combined deletions of the R C M (pufM) and RC (pu/L) genes (as described above), in pATSHR. This plasmid also contains the RC H (puhA) gene.  36  2.3 Construction of the if. sphaeroides APUHAAPUC strain The R. sphaeroides RC" L H 1 LH2" mutant strain APUHAAPUC was constructed +  through the chromosomal deletion of the pucBA genes (which express the LH2 a and (3 proteins) of the RC" A P U H A strain which contains a translationally in-frame (non-polar) deletion of the puhA gene (RC H protein) (12), using the suicide plasmid pSUP203 (which encodes resistance to ampicillin, tetracycline and chloramphenicol (63)) containing a 3.2-kb Pst I D N A fragment (705 bp of the pucBA genes were replaced with the neo gene (37)). The suicide plasmid pSUP203::705BA was conjugated into the A P U H A strain, and heterogenotes arising from a single homologous recombination event were selected on the basis of kanamycin resistance. Clones were grown aerobically in L B medium containing kanamycin for 48 hrs, after which various dilutions were spread onto L B plates containing kanamycin. Clones arising from a second homologous recombination event were selected on the basis of kanamycin resistance, and tetracycline sensitivity. The chromosomal D N A of the resultant colonies was purified and screened using PCR, and Southern blotting to identify the desired mutant strain.  2.4 Construction of the R. sphaeroides A R C L H strain The R. sphaeroides RC" LH1" LH2" mutant strain A R C L H was constructed by step-wise introduction of chromosomal deletions into the puf (LH1 and R C M and RC L genes) and puc (LH2 genes) operons of the R C H" A P U H A strain which contains a translationally in-frame (non-polar) of the puhA gene (RC H protein) (12), using the suicide plasmid p N G H l (which encodes resistance to kanamycin and contains the sacB gene for sucrose counterselection (50)) containing disrupted cloned D N A sequences for  37 the regions of interest. The chromosomal puf operon deletion utilized a cloned 4.57-kb Pst I fragment (which was engineered to contain EcoR I sites adjacent to the native Pst I sites) to replace the BspE I to Bel I intervening sequences with the linkers 5'C C G G A G G A T A G C A T G A T C A T T - 3 ' and 3' - T C C T A T C G T A C T A G T A A C T A G - 5 ' , yielding a puf deletion extending from 13 bp 5' of the pufB start codon to 19 bp 3' of the pufX stop codon. The chromosomal puc operon deletion utilized a cloned 3.2-kb Pst I D N A fragment (705 bp of the pucBA genes were replaced with the neo gene (37)) that was digested with Kpn I and self-ligated, resulting in the removal of the neo gene. Suicide plasmids containing the deleted puf and puc genes (encoded in plasmids p N G H 1: :PUFDEL and p N G H l : : D E L P U C respectively) were conjugated into the A P U H A strain (12), and heterogenotes arising from a single homologous recombination event were selected on the basis of kanamycin resistance on solid R C V media aerobically in darkness. Clones were grown aerobically in R C V minimal medium supplemented with 15 pg/ml of biotin and lOmg/ml niacin for seven days, after which 20 pi were plated on this R C V medium supplemented with 15% (w/v) sucrose, 1% (w/v) Bactotryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCI and 0.5% (v/v) dimethylsulfoxide (50). Plates were incubated under anaerobic dark conditions for 10 days, after which the chromosomal D N A of the resultant colonies was purified and screened using PCR to identify the desired mutant strains.  2.5  In vitro D N A  techniques  Restriction endonuclease digestion, D N A ligation, agarose gel electrophoresis, transformation of E. coli and other recombinant D N A procedures were carried out as  38 described (60). E. coli was transformed using the CaCb competent cell transformation procedure (60), or through electro-transformation, using a Gene Pulser apparatus according to the manufacturer's instruction manual (Bio-Rad Laboratories, Richmond, CA). Plasmid D N A was routinely isolated from E. coli or R. sphaeroides cultures using the QIAprep® Spin Miniprep Kit (QIAGEN Inc., Chatsworth, C A ) . D N A was purified from agarose gel slices by using the QIAEX® II Gel extraction kit (QIAGEN Inc., Chatsworth, CA). When necessary, D N A was purified from PCR reactions using the QIAquick® PCR Purification kit. D N A sequencing was performed by either the U B C N A P S facility, or by Sadowsky laboratory (Department of Biochemistry & Molecular Biology, UBC) using purified plasmid templates.  2.6 Bacterial conjugation The conjugation of plasmid D N A into R. sphaeroides strains was accomplished by use of E. coli DH10B donor strain, and the E. coli helper strain HB101(pRK2013) in tri-parental conjugations. Sometimes, E. coli strain SI7-1 was used instead. Recipient and donor cultures were grown to stationary phase, mixed at a 4:1 recipient: donor ratio (typically 400 p i : 100 pi), and pelleted (30 seconds, 15,000 x g in a benchtop microcentrifuge), and resuspended in 50 pi of R C V medium. A 10 p.1 portion of the suspension was spotted onto a R C V plate, and once the spot had dried, the plate was incubated at 30 °C overnight. Following the incubation period, a portion of the spot was resuspended in 1 ml R C V medium, and 100-350 pi were spread onto R C V plates with appropriate antibiotic(s). These plates were incubated at 30 °C, typically for 3-5  39 days, and R. sphaeroides exconjugants were purified from E. coli by spreading a single colony onto an L B plate containing appropriate antibiotic(s).  2.7 Absorption spectroscopy of intact cells Cells of semi-aerobic cultures grown to -150 Klett units (-72 hours) were pelleted, resuspended in 0.25 ml of R C V medium, and mixed with 0.75 ml of a 30% bovine serum albumin solution to reduce light-scattering. Spectral data were obtained on a TIDAS II spectrophotometer (World Precision Instruments) and analyzed using the J & M Spectralys program, version 1.82. Samples were scanned for light absorption over light wavelengths ranging from 350 nm to 1000 nm. Spectra were adjusted to the same 650 nm value (A=0.2), due solely to light-scattering of suspended cells, to compensate for minor differences in concentrations.  2.8 Flash spectroscopy Measurements were performed by my collaborator, Roger Prince at Exxon Mobile Research and Engineering Laboratories, using a double-beam spectrophotometer, as previously described (59). Chromatophore suspensions ( A  8 7 0  = 2.0) were reduced with  5 m M sodium ascorbate and allowed to equilibrate in the dark for at least 30 minutes before use, poising the ambient redox potential around E = +150 mV. The carotenoid h  bandshift (28) was measured at 490 minus 475 nm after a single actinic flash (10 ps full width at half height, filtered through a Wratten 88 A filter). The R C concentration was determined by the absorbance change at 605 minus 540 nm after a train of eight flashes separated by 32 ms, in the presence of 4 u M valinomycin and 1 u M antimycin (21).  40  2.9 Kinetics of RC formation Cultures of each strain were grown under high aeration (100 ml in 500 ml flasks shaken at 300 rpm) and then used as inocula for 800 ml cultures in 1 L flasks grown under low aeration. Samples of 1.5 ml were removed at time points following the switch to semiaerobic growth and analyzed using absorption spectroscopy as outlined above. The amounts of RC complex were determined by calculating the area underneath the corresponding 804 nm peak, and plotted as a function of time.  2.10 Electron transfer and proton uptake measurements Electron transfer and proton uptake measurements were performed by my collaborators, Pia Adelroth and Mark L. Paddock at the University of California San Diego, as previously described in (2, 56).  2.11 Chromatophore and soluble fraction preparations Cells from semi-aerobic R. sphaeroides cultures (or B. viridis grown photosynthetically) were pelleted, resuspended in 50 m M Tris-HCI buffer (pH 8.0), disrupted using a French pressure cell, and centrifuged at 25,800 x g for 10 min to obtain a supernatant liquid containing soluble proteins and suspended membrane vesicles (chromatophores); 3 ml of this supernatant were centrifuged for 14 min at 412,000 g, to pellet chromatophores; the supernatant liquid was collected and the chromatophores were resuspended in 3 ml of 50 m M Tris-HCI buffer, and the two fractions were centrifuged a second time for 14 min at 412,000 g; the chromatophore pellet was resuspended in 3 ml  41 of 50 m M Tris-HCI buffer to obtain the membrane fraction, and the top 0.35 ml of the supernatant sample were collected to obtain the soluble fraction. The B. viridis RC was purified as described (45), and the R. sphaeroides purified R C sample was a generous gift from M . Paddock (University of California, San Diego). Samples were stored on ice for flash spectroscopy or at -80 °C for western blots.  2.12 SDS-Gel electrophoresis and western blotting A glycine-SDS polyacrylamide gel (SDS-PAGE) system was used for electrophoresis of purified chromatophores and soluble fractions (61). Chromatophores (10 pg of protein as determined by a modified Lowry method (57)) were mixed with SDS-PAGE sample buffer (35), and heated at 95 °C for exactly 1 minute. Volumes of the soluble proteins proportional to the chromatophores in unfractionated extracts, and empirically determined amounts of pure RCs, were treated the same way. Samples were run on SDS-PAGE (12% acrylamide) at 100 volts for approximately 75 minutes. Gels were either stained using a Coomassie blue solution, or transferred onto P R O T R A N pure nitrocellulose membranes (Schleicher & Schuell, NH) for approximately 90 minutes at 100 volts. The R C H, R C M , or RC L proteins were detected, all as recommended in the E C L western blotting kit (Amersham Biosciences). The rabbit antisera raised against purified R. sphaeroides RC proteins were gifts, with the anti-RC H serum kindly provided by S. Kaplan (University of Texas) and the anti-RC M / L sera provided by E. Abresch (University of California, San Diego). The primary antibody was used at a 1:10,000 dilution, while the secondary antibody was used at 1:3000 dilution.  Routinely,  the incubation with the primary antibody was carried out overnight at 4 °C, whereas the  42 incubation with the secondary (HRP-labelled; see Abbreviations) was for 1 hour at room temperature. When necessary, the membranes were stripped of primary and secondary antibodies, through multiple (2 to 5) 10 minute washes with a Tris-buffered saline (TBS) solution (pH 2.0), followed by a single 15 minute incubation using a TBS-Tween (0.1% Tween-20™) solution (pH 7.6).  43  3. RESULTS  3.1 Studies of R C H protein domain swaps on photosynthesis and R C assembly in R. sphaeroides  3.1.1 Summary Initial in vivo experiments indicated that the RC H protein of B. viridis does not functionally substitute for the R. sphaeroides R C H protein. In attempts to identify specific regions of RC H that were responsible for this species specificity, the three domains of the R. sphaeroides R C H protein were replaced with the corresponding segments from the B. viridis protein. The effects of the mutations were evaluated in terms of photosynthetic growth, steady-state absorption spectra, light-driven electron and proton transfer reactions, western blot analyses of RC proteins in cellular membrane and soluble fractions, and kinetics of RC formation.  3.1.2 Development of the experimental system Plasmid p A T V H R was used to express the B. viridis R C H gene in R. sphaeroides A P U H A (LH2 ) and APUHAAPUC (LH2) strains, which contain a translationally in+  frame chromosomal deletion of the RC H gene (see (12); and Material and Methods section 2.3). The complemented strains were incapable of photosynthetic growth, although expression of the B. viridis RC H gene was indicated by absorption spectra of intact cells. For example, although the 760 and 804 nm RC peaks were absent from A P U H A A P U C (pATVHR) cells (Fig 3.1 A.), the LH1 870 nm peak was greater than that  44 of the strain that contained the expression plasmid pATP19P (which lacks a RC H gene; Fig. 2.2), and the broad region of absorbance centred at 770 nm indicates protein-free BChl and/or degradation products. The production of the B. viridis RC H protein in R. sphaeroides was also evaluated in western blot experiments, using the R. sphaeroides R C H antiserum (Fig. 3.IB). This antiserum reacted weakly with the RC H protein in purified B. viridis R C or chromatophore samples, but a band that was absent from the strain that contained pATP19P was obtained using chromatophores from the A P U H A A P U C (pATVHR) strain. I attribute this band to the B. viridis RC H protein, which migrated to a slightly lower M W position than in the B. viridis samples, and so perhaps the B. viridis RC H protein is proteolytically cleaved when present in R. sphaeroides. The expression of the B. viridis RC H gene in R. sphaeroides, coupled with the absence of a functional R C complex, provided the basis of a system that I used to exchange segments of these two structurally well-characterized R C H proteins, to investigate R C H protein domain-specific functions (Fig 3.2B). These experiments on hybrid R C H proteins are described in the following section.  45  B  1  2 3 4  5  6  Figure 3.1 A Absorption spectra of APUHAAPUC (LH2") intact cells containing plasmids: top, pATSHR (R. sphaeroides RC H gene); middle, p A T V H R (B. viridis RC H gene); bottom, pATP19P (expression vector lacking a RC H gene). Arrows point tothe 804 nm R C , and 870 nm L H 1 peaks. The vertical bar indicates A=0.15. B Western blot probed with R. sphaeroides RC H antiserum. Samples used were: lane 1, purified R. sphaeroides R C (control); lane 2, chromatophores from APUHAAPUC(pATSHR); lane 3, chromatophores from APUHAAPUC(pATP19P); lane 4, purified B. viridis RC; lane 5, chromatophores from B. viridis; lane 6, chromatophores from APUHAAPUC(pATVHR).  46 3.1.3 Overview of the hybrid RC H proteins To evaluate the function of the R C H protein periplasmic domain, a hybrid puhA (RC H) gene was constructed in plasmid pEVtip that encodes the B. viridis periplasmic amino acids in place of the corresponding R. sphaeroides sequence (see shaded residues in Fig. 3.2A). The R C H protein transmembrane domain was studied using a hybrid puhA gene constructed in plasmid pEVtrans, which encodes the B. viridis transmembrane domain (see underlined residues in Fig. 3.2A) flanked by the R. sphaeroides periplasmic and cytoplasmic domains. The resultant hybrid R C H protein contains the N-terminal 11 amino acids of R. sphaeroides followed by 23 B. viridis amino acids (Ile-12 to Arg-34) in place of the 22 R. sphaeroides amino acids (Leu-12 to Thr-33) that include the transmembrane a helices. This substitution is followed by the remaining 227 R. sphaeroides  C-terminal amino acids that form the bulk of the R. sphaeroides protein in a  globular, cytoplasmic domain (36). The periplasmic and transmembrane domain substitutions described above were combined in plasmid pEVPSCB (Fig 3.2B) to study the effects of replacing these two domains simultaneously, as opposed to individually. Thus, pEVPSCB encodes 34 B. viridis  residues from the N-terminal Met to Arg-34 in place of the 33 N-terminal R.  sphaeroides residues.  The effects of a R C H protein cytoplasmic domain substitution were studied using a hybrid R C H gene constructed in plasmid pEVCSP (Fig 3.2B), which encodes B. viridis cytoplasmic residues (starting at Glu-35) in place of the R. sphaeroides sequence that begins with Glu-34.  47  A  R.sphaeroides MVGVTAFGNFD' L A S L A I Y S F W I F L A G L I Y Y LQTENMR B.viridis MYHGALAQKLD IAQLVWYAQ W L V I W T W L L YLRREDR  37  37  B p  \  T C  0=  pATSHR wild type  pEVtip  pEVtrans  \ pEVPSCB  j  [  pEVCSP  Figure 3.2 A Amino acid alignment of R. sphaeroides and B. viridis R C H N terminal sequences. Shaded area indicates periplasmic amino acids, and underlined residues form an a-helix that spans the membrane. Identical amino acids are in bold, and the number 37 refers to the conserved R residue. Asterisks indicate R. sphaeroides residues changed by site-directed mutagenesis (see section 3.2). B Representation of the R C H proteins (with respective plasmid names provided below each) used in this study. P = periplasmic domain; T = transmembrane domain; C = cytoplasmic domain. Thick lines indicate B. viridis sequences.  48  3.1.4 Photosynthetic growth of LH2" APUHAAPUC strains expressing hybrid RC H genes The R. sphaeroides strain APUHAAPUC was used in these experiments to allow comparison of growth properties with the RC and LH1 levels measured in steady-state absorption spectra (see below). Cultures grown under semi-aerobic dark conditions (to induce the production of photosynthetic pigment-protein complexes) were used to inoculate media that were incubated under anaerobic illuminated (photosynthetic) conditions, with growth monitored over time. Although host cells containing pEVtip were photosynthetically incompetent, the replacement of the transmembrane domain (pEVtrans) supported photosynthetic growth, although to a lower final cell density than the wild type RC H-complemented strain (Fig. 3.3). The strain APUHAAPUC (pEVPSCB), which encodes a RC H protein consisting of both the periplasmic and transmembrane domains of B. viridis RC H in place of R. sphaeroides sequences, showed weak photosynthetic growth (Fig. 3.3). This growth appeared to be intermediate between the growth obtained when only the transmembrane domain was exchanged and the absence of photosynthetic growth when only the periplasmic domain was swapped. When the R. sphaeroides RC H cytoplasmic domain was replaced with the counterpart from B. viridis in pEVCSP, the resultant strain was incapable of photosynthetic growth (Fig. 3.3) The poor photosynthetic growth of APUHAAPUC strains complemented with mutant RC H genes could have been due to either low levels of the RC formed, or to weak catalytic activity of cells replete in a hybrid RC.  49  1000  n  10 4 0  ,  ,  50  100  Time (hrs)  Figure 3.3 Photosynthetic growth of R. sphaeroides APUHAAPUC (LH2") cells containing plasmids: pATSHR (wild type RC H , open circles); pEVtip (hybrid RC H containing B. viridis periplasmic domain, open triangles); pEVtrans (hybrid RC H containing B. viridis transmembrane domain, filled triangles); pEVPSCB (hybrid RC H containing B. viridis periplasmic and transmembrane domains, crosses); pEVCSP (hybrid RC H containing B. viridis cytoplasmic domain, asterisks). Ku, Klett units.  50  3.1.5 Absorption spectroscopy of LH2" APUHAAPUC strains expressing hybrid RC H genes To gain a better understanding of the reasons for the different photosynthetic growth properties of the strains described above, the absorption spectra of intact cells were recorded after semi-aerobic growth to induce the synthesis of pigment-protein complexes. The R. sphaeroides (LH2") APUHAAPUC strain was chosen because it is deficient in the production of LH2, thus revealing the positions and amplitudes of R C and LH1 peaks that are normally masked by the LH2 peaks. The primary data are presented in Figure 3.4, where the cellular content of the RC is indicated by the 804 nm peak amplitude, and are summarized in Table 3.1. These steady-state absorption spectra were normalized using the 650 nm value, which is due solely to light-scattering by suspended cells. Exchange of the periplasmic domain (pEVtip) reduced the amount of RC to 21% of the wild type amount. The substitution of the transmembrane domain (encoded by pEVtrans) reduced the amount of RC to 65% of the wild type, and when the periplasmic and the transmembrane domains were simultaneously replaced (pEVPSCB), there was a reduction in the amount of RC to 66% of the wild type. The substitution of the cytoplasmic domain (pEVCSP) resulted in a very low amount of the RC, too little for quantification using this method. The amplitude of the 870 nm LH1 peak was slightly reduced in the pEVtrans-containing cells, and slightly increased in the strain that contained pEVPSCB (Fig. 3.4). Although the amounts of R C indicated by the 804 nm peak area roughly correlate with the photosynthetic growth properties of strains lacking the LH2 complex, it was  conceivable that the photosynthetic growth of LH2 strains that contained these mutant R C H proteins would be different, and that the 804 nm peak area does not necessarily indicate the amount of catalytically active RC. Therefore I turned to an L H 2  +  background to evaluate photosynthetic growth, and used flash spectroscopy to measure the amount of R C and light-driven RC catalytic activity.  52  pATSHR pATP19P  pEVPSCB  pEVCSP  700  800  900 nm  Figure 3.4 Absorption spectra of APUHAAPUC (LH2~) intact cells containing plasmids: pATSHR (R. sphaeroides R C H); pATP19P (expression vector); pEVtip (hybrid R C H containing B. viridis periplasmic domain); pEVtrans (hybrid R C H containing B. viridis transmembrane domain); pEVPSCB (hybrid R C H containing B. viridis periplasmic and transmembrane domains); pEVCSP (hybrid R C H containing B. viridis cytoplasmic domain). The vertical bar indicates A=0.15.  53 3.1.6 Photosynthetic growth and flash spectroscopy of A P U H A strains expressing hybrid R C H genes The relative photosynthetic growth of the RC H mutants in the L H 2 background +  was similar to the growth of the LH2" strains, except that the periplasmic domain substitution (pEVtip) supported the growth of L H 2 cells (Fig. 3.5). +  For flash spectroscopy, cultures were grown semiaerobically, cells disrupted and membrane vesicles (chromatophores) were isolated. The percentage amount of the R C complex in mutants relative to the wild type as indicated by photobleaching (values were normalized to total BChl content) are given in Table 3.1 . This photobleaching technique measured the oxidation of the RC "special pair" (D) (see Materials and Methods). The values obtained are consistent with the photosynthetic growth properties and the steadystate absorption spectra of intact LH2" cells (see above). The carotenoid bandshift of chromatophores is due to changes in the absorption maxima of carotenoids when an electrochemical potential forms across the chromatophore membrane (28). In our experiments this phenomenon results from lightdependent electron and proton transfer reactions of the RC and cytochrome b/ci complexes (38). The carotenoid bandshifts of RC H mutants (relative to the wild type) were similar to the relative RC contents detected by photobleaching and absorbance spectra (Table 3.1). Taken together, the above data indicate that the impairments of photosynthetic growth in these R C H mutants is due to reductions in RC amounts, as opposed to effects on RC catalytic activity, with cellular photosynthetic energy transduction proportional to the amount of RC. This appears to be the case in either the presence or absence of LH2,  54 although the presence of LH2 can partially compensate for a reduced amount of R C when the reduction is not severe (i.e., >20% of the wild type, as in pEVtip). However, it was not clear whether the reduced R C content was due to a low amount of mutant R C H proteins in cell membranes, resulting in a commensurate reduction in the amount of the R C complex, or if membranes were replete in mutant R C H proteins that were impaired in the assembly of a stable R C complex.  T A B L E 3.1. Summary of R C levels and catalytic activities measured in steadystate absorption spectra of intact LH2" cells and flash spectroscopy of chromatophores obtained from L H 2 cells. See figure 3.2B on p. 47 for the +  phenotype of the various constructions.  Plasmid  % 804 nm  %RC  % crt bandshift  present in  peak area  LH2  LH2  L H 2 or LH2-  LH2" cells  chromatophores  +  3  +  b  +  chromatophores  b  strains pATSHR (wt)  100(1)  100  100  pEVtip  21(2)  24  24  pEVtrans  65 (3)  66  66  pEVPSCB  66(1)  ND  pEVCSP  U  d  C  18  ND 16  mean of experiments on cells from three independent cultures, with standard deviations in parentheses results of one experiment, with variation typically -10% not done undetectable  a  b  c d  1000  n  3  (A C  0)  100  Q  Q i_) 3 3  u  50  100  Time (hrs)  Figure 3.5 Photosynthetic growth of R. sphaeroides A P U H A (LH2 ) cells containing plasmids: pATSHR (wild type R C H , open circles); pEVtip (hybrid RC H containing B. viridis periplasmic domain, open triangles); pEVCSP (hybrid R C H containing B. viridis cytoplasmic domain, asterisks). K u , Klett units. +  56  3.1.7 RC H, RC M , and RC L protein levels in membrane and soluble cell fractions Cells of the A P U H A A P U C strain containing relevant plasmids were purified and probed in western blots with antisera directed against RC H, RC M or RC L proteins. The amounts of membrane and soluble fractions used were proportional to the nonfractionated cell extract, and exposure times were chosen to yield the same intensity of a constant amount of a purified RC control, to allow semi-quantitative comparisons of the amounts of these three proteins in the two fractions. As shown in Figure 3.6, the R. sphaeroides 28 kD band detected by the RC H antiserum was present in membranes of cells containing the wild type RC H gene (pATSHR) whereas the amount of RC H in the soluble fraction was extremely low. This band was absent from membrane and soluble fractions of the R C H" strain containing pATP19P. The amount of RC M in membranes of the RC H" strain was about the same as in the wild type R C H-complemented strain although, significantly, the amount of membrane-associated RC L was greatly reduced in the absence of the RC H protein. Strikingly, I discovered substantial amounts of RC M and R C L in the soluble fraction of cells that contained the wild type RC H protein in the membrane, whereas R C M and RC L were not detected in the soluble fraction of R C H" cells. These data indicate that the membrane-associated amount of RC M is independent of the presence of RC H . In contrast, membrane localization of RC L appears to require R C H in the membrane. Furthermore, the presence of substantial amounts of RC M and L in the cytoplasm correlates with the presence of RC H in the membrane.  Membrane preparations of cells expressing the RC H periplasmic domain substitution (pEVtip) contained less RC H than the wild type control, about the same amount of RC M , but less of RC L . The amounts of RC M and RC L in the soluble fraction were lower than in the wild type RC H control (pATSHR). Membrane and soluble fractions of cells containing the R C H transmembrane domain substitution (pEVtrans) contained about the same amounts of R C H , RC M and R C L as the wild type control, as did samples of cells containing the simultaneous exchange of periplasmic and transmembrane domains (pEVPSCB). The membranes of cells that produced the cytoplasmic domain-substituted RC H (pEVCSP) appeared to contain an extremely low amount of this mutant protein in western blots probed with the R. sphaeroides RC H antiserum. However, it is likely that this polyclonal antiserum reacts predominantly with cytoplasmic domain residues, which in pEVSCP would give a weak signal (as in the experiments on the B. viridis preparations; Fig. 3.1). I suggest that the amount of this hybrid RC H protein in membranes of R. sphaeroides, although low, is greater than indicated by the band intensity in Fig. 3.6. The amount of RC M was high whereas the amount of RC L was low in the membrane fraction, and the amounts of RC L and R C M in the soluble fraction were greatly reduced.  58  M  S  M  S !  M  S  ; M  S  :  H  mm  • mm PRC  pATSHR  mm  •  S  M  S  ••  mm mm  •  pATP19P  M  pEVtip  | pEVtrans  mm pEVPSCB  pEVCSP  3.6 Western blot probed with R. sphaeroides R C H , R C M or R C L antibodies. Samples probed were the R. sphaeroides purified RC (PRC), and membrane (M) and soluble (S) fractions of APUHAAPUC cells containing plasmids: pATSHR (R. sphaeroides RC H); pATP19P (no R C H gene); pEVtip (hybrid R C H containing B. viridis periplasmic domain); pEVtrans (hybrid RC H containing B. viridis transmembrane domain); pEVPSCB (hybrid RC H containing B. viridis periplasmic and transmembrane domains); pEVCSP (hybrid R C H containing B. viridis cytoplasmic domain). Figure  3.1.8 Kinetics of RC formation in the APUHAAPUC (pEVtrans) strain Absorption spectra of A P U H A A P U C (pEVtrans) cells, which express a hybrid puhA gene having the R. sphaeroides transmembrane segment replaced with the corresponding B. viridis sequence, revealed a membrane RC complex content lower than that of the wild type control (Fig 3.4), reduced the photosynthetic growth rate (Fig 3.3) and yielded RC complex catalytic activity that was 66% of the wild type control (Table 3.1). However individual R C protein amounts in the membrane appeared to be unaffected (Fig 3.6). To further investigate this interesting mutant, I determined the rate of formation and amounts of the RC complex in induction experiments. This was done by comparing the RC 804 nm peak area of APUHAAPUC cells complemented with either pATSHR (wild type puhA) or pEVtrans initially grown under high aeration (where synthesis of photosynthetic complexes are repressed), followed by a switch to low aeration to induce the production of photosynthetic complexes. In these experiments, the concentration of oxygen is initially high but, as cell numbers increase, the respiration of dissolved oxygen becomes greater than the diffusion of oxygen from the air. Thus, after about 15 to 20 hours of incubation, cells become starved for oxygen, growth slows, and the synthesis of the photosynthetic apparatus is induced (48). As shown in Figure 3.7A, the APUHAAPUC(pATSHR) and APUHAAPUC(pEVtrans) cultures grew identically under semi-aerobic conditions, but the kinetics of RC formation were quite different (Fig. 3.7B). Each data point in Figure 3.7 represents the calculated area under the corresponding peak at a time point after the initiation of low aeration (see Materials and Methods). Based on the results of several  60 experiments, the replacement of the R C H transmembrane domain reproducibly caused a slower initial rate of formation and lower final levels of the RC.  3  o  20  40  60  80  60  80  Time (hrs) B  0  20  40  Time (hrs)  Figure 3.7. Kinetics of RC formation. A Semi-aerobic growth curves for R. sphaeroides A P U H A A P U C cells complemented with either pATSHR (wild type puhA, filled diamonds) or pEVtrans (hybrid puhA open diamonds). B Area under R C 804 nm peak, normalized to light scattering at 650nm, and plotted as a function of time; wild type puhA (encoded in plasmid pATSHR), filled diamonds; hybrid puhA (encoded in plasmid pEVtrans), open diamonds. See Appendix 2 for absorption spectra at various time points.  61  3.2 Site-specific mutagenesis and deletion of the R. sphaeroides RC H protein domain segments  3.2.1 Summary This section details the results of experiments in which selected residues or short segments within the periplasmic or cytoplasmic domains of the R. sphaeroides R C H protein were replaced or deleted. The mutants of the R C H periplasmic domain were evaluated in terms of photosynthetic growth, steady-state absorption spectra, and western blot analyses of R C proteins in cellular membrane and soluble fractions. The mutants of the R C H cytoplasmic domain were evaluated in terms of photosynthetic growth, steady-state absorption spectra, light-driven proton translocation and electron transfer reactions.  3.2.2 Overview of the mutant RC H proteins The domain swapping experiments described in section 3.1 indicated that the B. viridis periplasmic domain substitution reduced the amount of the R C complex in cells to 21-24% of the wild type control (Table 3.1). This amount of RC was incapable of supporting photosynthetic growth in the absence of LH2 (Fig. 3.3), and resulted in slowed growth in the presence of LH2 (Fig. 3.5). It was thought that certain key residues present in the R. sphaeroides R C H (but differing in the B. viridis R C H) periplasmic domain might be important for RC complex assembly and hence photosynthetic growth.  62 The R C H periplasmic amino acids Thr-5 and Asn-9 were individually or together replaced with Vai and Leu, respectively (encoded in plasmids pEStiplMT, pEStiplMA, and pEStip2M). Residues Thr-5 and Asn-9 were chosen because they are the only amino acids in the periplasmic domain with polar side chains capable of forming hydrogen bonds with amino acids of other proteins. Additionally, codons 3 through 10 of the R. sphaeroides R C H periplasmic domain were removed in plasmid pEAPeri, to yield a mutant R C H protein in which Val-2 is followed by Asp-11 (see Fig. 3.2). I also decided to investigate R C H cytoplasmic surface residues that had been implicated as being near the entry point for one of three speculated proton transfer pathways into the R C from the cytoplasm, because of the inhibitory effects of Z n / C d 2 +  2+  binding to this region (see Introduction section 1.6). The R C H cytoplasmic residues His126 and His-128 were individually or together substituted with alanines (encoded in plasmids pEH126A, pEH128A, and p2XHis), to investigate their role in the uptake of protons from the cytosol.  3.2.3 Photosynthetic growth of APUHAAPUC strains expressing RC H periplasmic domain mutations The LH2" strain APUHAAPUC was used in these experiments to allow comparison of growth properties with the R C and LH1 levels indicated by absorption spectroscopy of intact cells. The APUHAAPUC (pEAPeri) strain was incapable of photosynthetic growth, while the single amino acid changes to the R. sphaeroides periplasmic domain (Thr5->Vai in pEStiplMT, and Asn-9->Leu in pEStiplMA) had no significant effects on  63 photosynthetic growth relative to the wild type (Fig. 3.8). In contrast, the mutant R C H gene encoding these two amino acid changes together in the plasmid pEStip2M resulted in slower growth that reached a plateau at a much lower final cell density than the control (Fig. 3.8; compare open squares to open circles).  3.2.4 Absorption spectroscopy of LH2" APUHAAPUC strains expressing RC H periplasmic domain mutations The individual changes of the periplasmic amino acids (Thr-5->Val in pEStiplMT, and A s n - 9 ^ L e u in pEStiplMA) did not result in significant changes to R C levels, whereas simultaneous changes of both amino acids (pEStip2M) decreased the R C level to 61% of the wild type (Fig. 3.9). No R C was detected in cells that produced a R C H protein from which most of the periplasmic domain had been deleted (pEAPeri) and, interestingly, there was a significant reduction in the 870 nm LH1 peak (Fig 3.9). It is noteworthy that the amplitude of the 870 nm LH1 peak was greater in cells that lacked an R C H protein (see pATP19P in Fig. 3.9) than in cells that produced an H protein from which amino acid residues 3 to 10 of the periplasmic domain had been deleted (encoded in plasmid pEAPeri). Thus, the cellular content of LH1 is affected more by the presence or absence of the R C H protein periplasmic domain, than by changes in periplasmic domain amino acid composition, or by the presence or absence of the wild type R C H protein.  64  1000 -!  Figure 3.8 Photosynthetic growth of R. sphaeroides APUHAAPUC (LH2~) cells containing plasmids: pATSHR (wild type RC H, open circles); pEAPeri (mutant RC H lacking the periplasmic domain, filled squares); pEStiplMT (mutant RC H with the periplasmic residue Thr-5 replaced with Vai, filled diamonds); pEStiplMA (mutant RC H with the periplasmic residue Asn-9 replaced with Leu, open diamonds); or pEStip2M (mutant RC H with both periplasmic residues Thr-5 and Asn-9 replaced with Vai and Leu, respectively; open squares).  65  pATSHR pATP19P  pEStiplMT  pEStiplMA pEStip2M  pEAPeri  700  800  900 nm  Figure 3.9 Absorption spectra of APUHAAPUC (LH2") intact cells containing plasmids: pATSHR (R. sphaeroides R C H); pATP19P (expression vector); pEVtip (hybrid R C H containing B. viridis periplasmic domain); pEStiplMT (mutant R C H with the periplasmic residue Thr-5 replaced by Vai); pEStiplMA (mutant R C H with the periplasmic residue Asn-9 replaced by Leu); pEStip2M (mutant R C H with periplasmic residues Thr-5 and Asn-9 replaced by Vai and Leu, respectively); or pEAPeri (mutant R C H lacking the periplasmic domain).  3.2.5 Flash spectroscopy of APUHA strains expressing RC H periplasmic domain mutations The percentage amounts of the R C complex in chromatophores from L H 2 cells +  containing these mutant periplasmic domains relative to the wild type, as indicated by photobleaching (due to electron transfer from D to QA) are given in Table 3.2. These values are consistent with the photosynthetic growth properties and the steady-state absorption spectra of LH2" cells (see above). The carotenoid bandshifts of RC H periplasmic domain mutants (relative to the wild type) were similar to the relative R C contents detected by photobleaching and absorbance spectra (Table 3.2).  T A B L E 3.2 Summary of RC levels and catalytic activities measured in steadystate absorption spectra of intact LH2" cells and flash spectroscopy of chromatophores obtained from L H 2 cells. +  Plasmid  % 804 nm  % RC  % crt bandshift  present in  peak area  LH2  LH2  L H 2 or LH2  L H 2 cells  chromatophores  chromatophores  100  100  +  -  3  +  15  +  15  strains pATSHR (wt) pEAPeri  100(1) U  c  ND  D  ND  pEStip2M  61(2)  69  89  pEStiplMA  89 (3)  100  118  pEStiplMT  91 (2)  100  116  mean of experiments on cells from three independent cultures, with standard deviations in parentheses results of one experiment, with variation typically -10% undetectable not done  a  b 0 d  67  3.2.6 RC H, RC M and RC L protein levels in membrane and soluble cell fractions of strains expressing RC H periplasmic domain mutationsCells of the A P U H A A P U C strain containing relevant plasmids were disrupted and fractions probed with antisera directed against RC H , R C M or RC L proteins in western blots. The amounts of membrane and soluble fractions used were proportional to the non-fractionated cell extract, and exposure times were chosen to yield the same intensity of a constant amount of a purified R C control, to allow semi-quantitative comparisons of the amounts of these three proteins in the two fractions. Membranes of cells containing the R C H periplasmic domain deletion (pEAPeri) contained a vanishingly small amount of this mutant R C H protein, the amount of R C M was equivalent to the wild type control (pATSHR), and the amount of R C L was very low. A l l three of the R C proteins were undetectable in the soluble fraction (Fig. 3.10). The R C H protein containing both periplasmic domain site-directed amino acid changes (pEStip2M) was present in membranes at a slightly lower level than that observed for the two single amino acid changes, with a slightly reduced amount of RC L , whereas R C M was present at the same level as in the wild type control. The amounts of RC M and R C L proteins in the soluble fraction were comparable to the wild type (Fig. 3.10). Membranes of cells expressing either of the single amino acid changes in the periplasmic domain of R C H (pEStiplMA and pEStiplMT) contained slightly decreased amounts of RC H , whereas the amounts of RC M and RC L were about the same as in the wild type control. The amounts of R C M and R C L in the soluble fractions did not differ greatly from the wild type control (Fig. 3.10).  68  M  s  mm  H  M  s  S  M  UH  S  Bl  PRC  pATSHR  pATP19P  Figure 3.10 Western blot probed with R.  pEAPeri  pEStip2M  M  S  mmm. !  |  S  mmm—  •Hi  Bit H i — i  L  M •  *m*mmmm  M  M  mmm pEStiplMT  pEStiplMA  sphaeroides R C H , R C M or R C L antibodies.  Samples probed were the R. sphaeroides purified RC (PRC), and membrane (M) and soluble (S) fractions of A P U H A A P U C cells containing plasmids: pATSHR (R. sphaeroides R C H); pATP19P (no R C H gene); pEAPeri (mutant RC H lacking the periplasmic domain); pEStip2M (mutant RC H with periplasmic residues Thr-5 and Asn9 replaced by Val and Leu, respectively); pEStiplMT (mutant R C H with the periplasmic residue Thr-5 replaced by Val); and pEStiplMA (mutant R C H with the periplasmic residue Asn-9 replaced with Leu).  69  3.2.7 Photosynthetic growth and absorption spectroscopy of APUHAAPUC strains expressing RC H cytoplasmic domain mutations Photosynthetic growth of the APUHAAPUC host cells expressing mutant RC H proteins with either single or double substitutions of His-126 and His-128 residues to alanines (encoded in plasmids pEH126A, pEH128A, and p2XHis respectively), closely paralleled that of the wild type control (pATSHR) (Fig. 3.11 A). As shown in Figure 3.1 IB, the absorption spectra of intact cells of APUHAAPUC strains containing pEH126A, pEH128A, or p2XHis plasmids revealed no significant differences between the RC and LH1 peaks of these strains (at 804 and 870 nm, respectively), and that of the wild type control (pATSHR). This is in agreement with the photosynthetic growth characteristics for these strains.  3.2.8 Electron transfer and proton uptake measurements for APUHAAPUC strains expressing RC H cytoplasmic domain mutations The translocation of protons into the RC for the protonation of the Q B quinone is coupled to two sequential light-induced electron transfer reactions (see Introduction section 1.6). To determine the role of R C H surface residues His-126 and His-128 in the delivery of protons into the RC, proton uptake rates and the coupled electron transfer rates were measured using RCs purified from A P U H A A P U C host cells containing mutant RC H proteins with individual or simultaneous replacements of the cytoplasmic residues His-126 and His-128 (encoded in plasmids pEH126A, pEH128A, and p2XHis respectively), to alanines. Cultures of these cells were grown semi-aerobically, cells disrupted and RCs purified from isolated membrane vesicles (chromatophores).  70  B  pATSHR  p2XHis  pEH126A pEH128A  900 nm  Figure 3.11 A Photosynthetic growth of R. sphaeroides A P U H A A P U C (LH2") cells containing plasmids: pATSHR (wild type R C H , open circles); p2XHis (His-126 and His 128 -» Ala, open triangles); pEH126A (His-126^ Ala, closed squares); pEH128A (His-128^ Ala, open diamonds). B Absorption spectra of A P U H A A P U C (LH2") intact cells containing select plasmids.  71 Electron transfer and proton uptake reactions in the R C were triggered by laser flashes (see Material and Methods section 2.10). The overall electron and proton transfer reactions catalyzed by the RC are summarized in Fig 1.7A. The first electron transfer reaction from Q to Q B , and the coupled proton uptake can be represented as: A  kAB  QA" + Q B + H +(Glu-L212)"  (eq. I)  Q A + QB" + (G1U-L212)H  +  Although Q B is not protonated in this step, it is converted into a quasi-stable anionic semiquinone following the acceptance of an electron from Q . This process also A  involves the protonation of the QB" proximal RC L acid residue Glu-212 (51). Following a single laser flash, the rate of proton uptake by Glu-212 for purified RCs of A P U H A A P U C host cells containing plasmids pEH126A, pEH128A, or p2XHis was measured by monitoring proton uptake from solution and the coupled electron transfer rate kAB • In comparison with the native RC, while the individual changes of the His 1  residues to Ala only slightly decreased the rate of proton uptake and kAB (data not shown), both were significantly decreased for RCs of APUHAAPUC(p2XHis) cells (Fig 3.12). For the native R C , the proton uptake rate and kAB were calculated to be -4000 s" . 1  In contrast, the rates for the double mutant was reduced to -500 s" . 1  The Q " semiquinone is converted to Q B H after a second flash, resulting in the B  2  uptake of a second proton from solution and transfer of a second electron from Q " ; this A  process is represented below.  QA" + QB" + H + (Glu-L212)H +  Q A + Q B H + (G1U-L212)" 2  (eq. 2)  72  Proton uptake  Q O o  < t  4 6 — time [ms]  First electron transfer &AB r  •  i  •  i  •  i  1  i  B.  Native Q O a  If  i  Double mutant  < j  0 •-  .  4 ; 6 ~~ time [ms]  i  10  Figure 3.12 Measurement of proton uptake (A), and electron transfer kAB (B) processes at pH 8.5 for purified RCs of native R. sphaeroides, and of double His mutant (His-126 and His-128 -» Ala change). Proton uptake was measured at 580 nm using the pH-sensitive dye m-cresol purple. Electron transfer was measured at 750 nm, which is sensitive to the reduction states of the quinones. These experiments were performed in collaboration with the Okamura laboratory (UCSD, Ca). 1  73 The transfer of a second electron to QB" is coupled to its protonation by R C L residue Ser223, which is rapidly re-protonated by proton uptake from the solvent, yielding the intermediate Q B H " . A second proton is internally transferred to Q B H " from Glu-212, which was protonated during the first electron transfer reaction to yield the quinol  QBH2  (51). Following a second laser flash, the rates of proton uptake by QB" for purified RCs of A P U H A A P U C host cells containing plasmids pEH126A, pEH128A, or p2XHis were measured by monitoring proton uptake from solution and the coupled electron transfer 1  2  rate k B (Fig. 3.14). These rates were calculated to be -350 s" for the native RC. No A  significant effects on either rate were observed for RCs of A P U H A A P U C cells containing either pEH126A, or pEH128A plasmids (data not shown). However, proton uptake and k B rates for the R C of the double mutant were decreased to -100 s" . 2  1  A  To investigate whether the changes in RC catalytic activity were due specifically to the loss of the His side chains (Fig. 3.13), as opposed to secondary effects such as protein conformational changes, exogenous proton donors such as imidazole were used to determine if they would rescue the observed effects on the proton uptake kinetics of A P U H A A P U C (p2XHis) strain. HjN*  CH  |  COO-  Imidazole side chain  Fig. 3.13. Chemical formula of the amino acid histidine.  74  Proton uptake  i—i—i—i—(—i—i—i—i—i—  0  5  10 15 20 25 30 35 40 time [ms]  Second electron transfer &AB 20  Q O  15  __ 10  o  <  c  5  t  0 -J—t—I—I—I—I—I—I  0  I  I  I  I  I  I  I  .  1_  5 10 15 20 25 30 35 40 —"time [ms]  Figure 3.14 Measurement of proton uptake (A), and electron transfer k B (B) processes at pH 8.5 for purified RCs of native R. sphaeroides, and of double His mutant (His-126 and His-128 -» Ala change). Proton uptake was measured at 580 nm using the pH-sensitive dye m-cresol purple. Electron transfer was measured at 450 nm, the peak absorption of the semiquinone species. The initial rise is due to the formation of the Q A QB" state, which decays to (QBH)" resulting in the observed transient. These experiments were performed in collaboration with the Okamura laboratory (UCSD, Ca). A  75 The addition of 50 m M imidazole to the double mutant RC sample increased the rate of the both electron transfer reactions to nearly equal to that observed for the native RCs (Fig. 3.15), with the effects being reversible upon the removal of imidazole (data not shown). Addition of imidazole had no effect on the electron transfer rates for the native, or single mutant RCs (data not shown).  First electron transfer AAB  0  2  4  6  8  10  12  time [ms] Second electron transfer &AB T— —i— —i— —i— —i— —i—•—i—•—i—•—r 1  0  1  5  1  1  1  10 15 20 25 30 35 40  — • time [ms]  Figure 3.15 "Chemical rescue" by exogenous imidazole of kAB (A) and kAB (B), for purified RCs of native R. sphaeroides, and of double His mutant (His-126 and His-128 -» Ala change). These experiments were performed in collaboration with the Okamura laboratory (UCSD, Ca). 1  2  76  3.3 RC gene deletion studies  3.3.1 Summary Possible interactions between photosynthetic reaction centre (RC) proteins were evaluated by use of R C gene-specific deletions. The RC H , R C M and RC L proteins were produced, either alone or in concert with one or both of the others, in a strain of R. sphaeroides that contained chromosomal deletions of all three RC genes (ARCLH strain). The properties of these strains were studied in photosynthetic growth and absorption spectroscopy experiments. The amounts of the RC H , RC M , and R C L proteins in cell membrane and soluble fractions were assessed in western blots.  3.3.2 Overview of the R. sphaeroides A R C L H strain and the RC gene deletions M y western blots showing RC H , R C M , and R C L levels in cells expressing mutant RC H genes appeared to be at odds with reigning models of RC complex assembly (see Results sections 3.1.7 and 3.2.6). This is because previous publications had proposed that the R C H protein is a "foundation" or "chaperone-like" protein that is the first protein to accumulate in the membrane for subsequent efficient assembly of RC M and RC L proteins to form the RC holocomplex. Furthermore, the level of R C M in R C H-deleted cells was reported to be low because of degradation (13, 68), whereas my experiments on RC H mutants indicated that the accumulation of RC M in the membrane was independent of RC H . Moreover, the accumulation of R C L had not been studied prior to my research. Therefore, I created a host strain and several plasmids to further  77 investigate the relationships between the R C H, R C M , and R C L proteins, in terms of their accumulation within the cell. The A R C L H mutant strain contains chromosomal deletions in the puh, puf and puc genes, yielding it incapable of producing the RC, LH1 or LH2 complexes. The plasmid pESHPUF, which encodes the native R. sphaeroides puhA and puf operon genes was constructed to assess the recovery of RC and LH1 production in the A R C L H strain to test the functionality of this strain and this plasmid for RC gene deletion studies. The deletion of the R C L gene was studied using plasmid pESHPUFL", which expresses the R C H and R C M genes. Similarly, plasmid pESHPUFM" was used to produce the R C H and RC L proteins in the absence of RC M . The effects of the deletion of the R C M and R C L genes on the R C H membrane content was investigated using plasmid pESHPUFL'M", with the converse (RC M and R C L produced without RC H) examined using plasmid pEPUF. A l l of the R C gene deletions in the complemented A R C L H strain were translationally in-frame deletions, to minimize the possibility of polar effects on other cotranscribed genes.  3.3.3 Photosynthetic growth of A R C L H strain expressing different combinations of RC genes The strain A R C L H containing the expression plasmid pATP19P was incapable of photosynthetic growth, as it was when containing pESHPUFL", pESHPUFM", pESHPUFL'M" or pEPUF (Fig. 3.16). In contrast, the A R C L H (pESHPUF) strain grew  78 almost as well as the wild type, indicating that the puhA (RC H) gene and the puf genes (LH 1 and R C L / M ) on plasmid pESHPUF were expressed at a level sufficient for photosynthetic growth.  1000  n  Figure 3.16 Photosynthetic growth of wild type R. sphaeroides strain 2.4.1 (filled triangles), and complemented A R C L H strains. Plasmids used to express R C genes in A R C L H were: pESHPUF (expressing R C H , R C M and R C L genes, open circles); pATP19P (expression vector, open triangles); pESHPUFL" (expressing RC H and R C M genes, open squares); pESHPUFM" (expressing R C H and RC L genes, asterisks); pESHPUFL'M" (expressing R C H gene, crosses); and pEPUF (expressing R C M and R C L genes, filled diamonds).  79  3.3.4 Absorption spectra of the A R C L H strain expressing different combinations of RC genes Absorption spectra of intact ARCLH(pATP19P) cells grown semi-aerobically showed that this strain is devoid of RC, LH1 and LH2 complexes, because of the absence of peaks in the 700 to 950 nm range (Fig. 3.17 ). The RC and LH1 complexes were restored by complementation of A R C L H with the plasmid pESHPUF, as indicated by the 760, 804 and 870 nm peaks (Fig. 3.17). The RC 760 and 804 nm peaks were absent from cells containing plasmids pESHPUFL", pESHPUFM", pESHPUFL'M" or pEPUF (Fig 3.17). There was a small 770 nm peak in A R C L H (pEPUF), indicative of free bacteriochlorophyll (40). There were also differences in the amplitude of the L H 1 870 nm peak in the different strains. I cannot readily account for these changes, except to attribute them to a variety of unknown interactions between LH1 and the R C (see Discussion section 4.3). These observations are consistent with the photosynthetic growth results, and together indicate that: 1) the RC and LH1 genes are expressed in the ARCLH(pESHPUF) strain, to produce a functional RC and L H 1 ; 2) the A R C L H strain provides a system to express different combinations of plasmid-borne RC genes for the study of potential interactions between R C proteins in membrane localization and accumulation.  pEPUF  pESHPUF  pESHPUFL"  pESHPUFM"  pESHPUFL"M" pATP19P 700  800  900 nm  Figure 3.17 Absorption spectra of A R C L H intact cells containing plasmids: pEPUF (encoding R C M and R C L), pESHPUF (encoding R C H , R C M , and R C L), pESHPUFL" (encoding R C H and R C M), pESHPUFM" (encoding R C H and R C L), pESHPUFL"M" (encoding RC H only), or pATP19P (empty expression vector).  81  3.3.5 RC H, RC M and RC L protein levels in membrane and soluble cell fractions Cultures of the A R C L H strain containing plasmids that express different combinations of RC H, RC M and RC L genes were grown semi-aerobically, cells disrupted, and the soluble and membrane fractions probed in western blots using antisera directed against J?. sphaeroides RC H , RC M or RC L proteins. The amounts of membrane and soluble fractions used were proportional to the non-fractionated cell extract, and exposure times were chosen to yield the same intensity of a constant amount of a purified RC control, for semi-quantitative comparisons of the amounts of these three proteins in the two fractions. As shown in Figure 3.18, the RC H, RC M and RC L proteins were detected in the membrane fraction of A R C L H cells complemented with the positive control plasmid pESHPUF, which was constructed to express all three RC proteins, whereas cells containing the negative control plasmid pATP19P lacked these bands. The RC M protein was detected in the membrane fraction of cells containing the RC M gene, regardless of the presence of the RC H or RC L genes. The RC H protein was detected in membranes only when both RC M and RC L proteins were present, whereas the membrane-associated RC L protein was detected only when the RC M protein was present (Fig. 3.18). However, the amount of RC L present in membranes containing RC M but not RC H was -27 % of the amount detected when both RC H and RC M were present. These data indicate that accumulation of RC H in the membrane requires the presence of both RC M and RC L. Insertion and amounts of RC L in the membrane is  82 low, but detectable in the absence of R C H , and is the greatest in the presence of both R C H and R C M . Steady-state levels of R C M in the membrane are independent of R C H and R C L . No R C H was found in any of the soluble fractions (Fig. 3.18). In contrast, R C M and R C L were readily detected in the soluble fraction of the positive control (pESHPUF), traces of R C M and R C L were present in the soluble fraction of R C H" (pEPUF) cells, and neither R C M nor RC L was detected when the other of these two proteins was absent from cells (Fig. 3.18). Thus, the accumulation of R C M and R C L in the soluble fraction is mutually dependent, and the presence of the R C H protein in the membrane appears to result in maximal amounts of soluble forms of R C M and R C L proteins destined for membrane insertion and R C assembly.  o. X  3 0-  OH  s00 00s OH  00  UJ  D  5  OH  OO  UH  UH  PH  D  u  —1  HJ  2 UH  W  CH  D CH UJ CH  UJ  a  LU O.  H  UH  M L II  H  H  M"  M  M"  L  i:  L"  II M L  H"  M L  B UH  HJ UH  X  OH  5 0. 00 UJ  CH  5 X  oo  HJ U-.  CH  00  UJ CH  uu  H M  H  L"  L  CH  X  oo  UJ CH  5 UH  OH  w CH  OH  H M L H M"  L  M"  H M L  H  M L  Figure 3.18 Western blots of cell fractions probed with R C H , RC M or R C L antibodies. A : membrane fraction. B : soluble fraction. Samples were of A R C H L H cells containing plasmids as indicated.  84  4. DISCUSSION  4.1 Functional analysis of if. sphaeroides RC H protein domains The structurally well characterized, but functionally poorly understood R. sphaeroides RC H protein can be conceptually divided into three domains: 1) a ~11 amino acid N-terminal domain located on the periplasmic side of the inner membrane 2) a single transmembrane a-helix; 3) a C-terminal, large, globular cytoplasmic domain (36). The function of each of these domains was investigated in domain substitution, deletion, and site-specific mutagenesis experiments. The domain substitution approach arose from genetic complementation experiments on R. sphaeroides RC H gene deletion strains that expressed the B. viridis R C H gene, but were incapable of photosynthetic growth. Although the B. viridis RC H protein could in principle have resulted in an equivalently low amount of RC formed in R. sphaeroides, insufficient to support photosynthetic growth or produce a detectable 804 nm RC peak in the absorption spectrum, the appearance of a broad peak centred at 770 nm (the wavelength of protein-free bacteriochlorophyll absorption (40)) (Fig. 3.1) suggests that the B. viridis RC H protein was incapable of interacting with the R. sphaeroides RC M and R C L proteins in RC complex assembly. As there were several ways to explain these results, I reduced the complexity of analysis by creating and using strains that contained only one or two RC H domain sequence changes, to investigate possible contributions of these individual domains to RC catalytic activity, assembly of the R C complex and membrane localization of RC proteins.  85 A l l of the R C H genes were expressed using the puc promoter (32) of pATP19P, and the R C M and RC L genes were expressed from the wild type chromosomal puf operon. Therefore reductions in the steady-state levels of RC proteins are attributed to proteolysis that occurs when these proteins fail to interact to form the R C complex (68). The periplasmic domain. Photosynthetic growth was impaired in LH2" R. sphaeroides cells that contained the periplasmic domain replacement, deletion or the double amino acid substitution, whereas the single amino acid changes did not greatly affect photosynthetic growth (Figs. 3.3 and 3.8). The absorption spectra in the LH2" background (Figs. 3.4 and 3.9) and the photobleaching experiments in the L H 2  +  background (Tables 3.1 and 3.2) indicate that, regardless of the presence of LH2, the amount of the R C complex in these strains is congruent with their photosynthetic growth properties. Because the carotenoid bandshift of these strains was approximately proportional to their R C content, changes in the periplasmic domain of R C H do not seem to affect light-driven electron transfer and proton translocation in the reduced amounts of RCs that were assembled. It appears that there is a minimal cellular R C content that supports photosynthetic growth under the conditions in this study (of growth medium, light intensity, etc), which is estimated to be in the range of 60 to 70% of the wild type amount when LH2 is absent, and 20 to 30% of the wild type amount when LH2 is present. The western blot results (Figs. 3.6 and 3.10) show the importance of the periplasmic domain for RC H levels in the membrane fraction, and reveal a correspondence between the amount of membrane-associated R C H and membraneassociated RC L , but not R C M . For example, the deletion of periplasmic residues 3 to  86 10 (pEAPeri) resulted in only a trace of RC H in the membrane fraction, similar to the complete absence of RC H . In both cases the amount of membrane-associated RC L was greatly reduced whereas the amount of R C M was about the same as in the wild type control. When the R. sphaeroides RC H periplasmic domain was replaced with the B. viridis sequence (pEVtip), the membrane-associated RC H and L proteins were detected at levels lower than the wild type control, while the amount of RC M in membranes was not reduced. The single substitutions in the periplasmic domain (Thr-5 -> Val in plasmid pEStiplMT, and Asn-9  Leu in pEStiplMA) had little effect on the amounts of R C H  and R C L in membranes, whereas the double substitution (pEStip2M) caused a greater reduction in the R C H and L levels. None of these mutations reduced the amount of membrane-associated RC M . Thus it appears that there are several amino acids in the periplasmic domain that together affect RC H (and hence RC L) amounts in the membrane, whereas the amount of membrane-associated RC M is independent of RC H and R C L . It was surprising to discover that in strains which produced the wild type RC H protein, and hence were replete in all three RC proteins, significant amounts of RC M and R C L (but little RC H) were detected in the soluble fraction of disrupted cells (Fig. 3.6). This result is counter-intuitive, because R C M and RC L are much more hydrophobic than R C H (i.e. R C M and RC L each contain five transmembrane oc-helices and relatively short extra-membranous segments, whereas RC H contains a single transmembrane a-helix and a larger cytoplasmic domain). Cells containing RC H proteins with periplasmic domain mutations that impaired membrane localization contained lower amounts of both RC M and RC L proteins in the soluble fraction.  87 Therefore, the presence of the R C H protein in the membrane, which is in part dependent on the periplasmic domain sequence, appears to be needed to sustain wild type levels of R C M and R C L proteins in the cytoplasm. I at first thought that upon membrane insertion, RC H might interact with one or more R C putative assembly factors (3, 12, 72), and that this interaction stabilizes newly translated RC M and R C L proteins in a soluble form for membrane insertion dedicated to R C assembly. However, the subsequent R C gene deletion experiments (see Results section 3.3.5; and Discussion section 4.2) point to membrane accumulation of RC L for accumulation of RC M and L in the cytoplasm. The transmembrane domain. In contrast to the periplasmic domain, substitution of the R. sphaeroides R C H transmembrane domain with the radically different and one amino acid longer B. viridis sequence (Fig. 3.2) encoded by pEVtrans did not appear to affect membrane localization of RC H or RC L, and R C M was again present at the wild type level (Fig. 3.6). The amounts of these proteins in the soluble fraction were also similar to the wild type control. Thus, in spite of a greatly different amino acid sequence, a stretch of 20 to 21 amino acids in the ot-helical transmembrane domain suffices for R C H and, consequently, R C L membrane insertion. However, there appears to be a secondary role of the R C H transmembrane domain in R C assembly. This is because although the substitution of the transmembrane domain did not affect the amounts of individual R C proteins, the initial rate of formation (Fig. 3.7) and steady-state level (Fig. 3.4 and Table 3.1) of the hybrid RC complex were lowered. I attribute these reductions to an inability of the B. viridis transmembrane sequence to properly interact with the R. sphaeroides R C M protein, thus resulting in impaired R C assembly and photosynthetic growth (Fig. 3.3).  88 Analysis of the R. sphaeroides  RC crystal structure (Brookhaven PDB accession  1 ALT) indicates that only amino acids of the RC M protein could interact with RC H transmembrane amino acid side chains. R C H transmembrane residues with atoms located <3.5 A of RC M residue atoms are (RC M amino acids given in parentheses): Ser-14 (Trp-297, His-301); Phe-20 (Leu-204); Tyr-30 (Arg-267); Gln-32 (Phe-258); Glu34 (Arg-267); Asn-35 (Gly-264, Ile-265, Trp-268). Several of these interactions appear to be hydrogen bonds (e.g.: RC H residues Ser-14, Tyr-30, Glu-34 and Asn-35) and so are candidates for determinants of quaternary structure in the R. sphaeroides  native RC.  A l l of these residues are changed in the transmembrane substitution (Fig. 3.2 A). The carotenoid bandshift of chromatophores containing the transmembrane substituted R C H was within experimental error proportional to the R C content (Table 3.1). Therefore, the significance of the R C H / M amino acid interactions discussed above appears to be solely in the assembly of the RC, with little or no effect on R C catalytic activity. Simultaneous  substitution  of periplasmic  above, substitution of the R. sphaeroides  and transmembrane  domains.  As noted  with the B. viridis periplasmic domain reduced  the amount of RC H in the membrane, whereas the analogous transmembrane substitution did not appear to affect membrane localization. These two domain changes were combined (in plasmid pEVPSCB), resulting in a RC H protein consisting of B. viridis sequences from the N-terminus to amino acid Arg-34, followed by the R.  sphaeroides  cytoplasmic domain (Fig. 3.2). The simultaneous substitution of periplasmic and transmembrane domains yielded cells with photosynthetic growth properties (Fig. 3.3), R C complex absorption and  89 activity (Fig. 3.4 and Table 3.1), and amounts of membrane-associated and soluble forms of R C H , R C M and R C L proteins (Fig. 3.6) that were more like those resulting from the transmembrane than the periplasmic individual substitutions. One way to view these results is that the inhibitory effect of the periplasmic substitution on the level of RC H in the membrane is ameliorated by the addition of the transmembrane substitution. I speculate that species-specific interactions between periplasmic and transmembrane residues enhance R C H insertion into the membrane. This speculation led to the creation of periplasmic domain deletion and site-directed mutants (see below). The cytoplasmic domain substitution. Photosynthetic growth was not observed for LH2" or L H 2 host cells that contained a substitution of the R. sphaeroides R C H 226 +  amino acid cytoplasmic domain with the B. viridis 223 amino acid domain (-40% identity; see Appendix 1) (Fig. 3.3). Absorption spectra (Fig. 3.4), flash spectroscopy (Table 3.1) and western blot experiments (Fig. 3.6) on strains containing plasmid pEVCSP indicate that the significant reductions in steady-state levels of the membrane R C content, and the commensurate loss of photosynthetic growth is the result of impaired R C complex assembly. As with the other domain mutations that reduced the amounts of membrane-associated RC H and R C L , and the cytoplasmic amounts of R C M and R C L , there was not a reduction of the amount of RC M in the membrane. Thus, the R C H cytoplasmic domain also functions to obtain an optimal amount of RC H in the cell membrane, which in turn is needed for accumulation R C L in the membrane, and subsequent accumulation of RC M and R C L proteins in the cytoplasm of for R C assembly.  90 The function of the cytoplasmic domain extends beyond an assembly role, as it was previously implicated in the translocation of protons from the cytoplasm to QB(53). The binding of C d  2 +  or Z n  2+  by R C H cytoplasmic residues Asp-124, His-126 and His-  128 was shown to result in a lowering of the Q B quinone protonation rate (5). In a collaborative effort with the Okamura laboratory, we determined that individual changes of R C H cytoplasmic surface residues His-126 and His-128 residue to Ala had insignificant effects on steady-state levels of the RC, or its. overall catalytic activity (Fig. • 3.11). However, the simultaneous changes of both His residues to Ala decreased proton uptake rates by 4-10 fold (Figs. 3.12 and 3.13). These reductions are attributed to a slower rate of proton delivery from the entry point to the proton transfer pathway, due to the absence of the His-126 and His-128 imidazole side chains. The addition of exogenous imidazole as a proton donor restored proton uptake rates to almost wild type levels (Fig. 3.15). Therefore, it can be concluded that His-126 and His-128 jointly mediate proton transfer into the RC by acting as proton donors at the entrance of the proton transfer pathway as reported in detail (2, 52), and outlined in Figure 4.1. These conclusions are confirmed by experiments described in (56).  91  Figure 4.1 Part of the structure of the R C in the charge-separated state. Shown are the Q B quinone, and amino acid residues thought to be key for the transfer of protons H (1), and H (2) to Q B . The proton path through R C H residues His-126 and His-128 to Q B are represented by a black dashed arrow for H (1), and a gray dashed arrow for H (2). Potential hydrogen bonds are indicated by small dashes, and water molecules revealed by x-ray crystallography (1) represented by large dots. This figure was kindly provided by Mark Paddock. +  +  +  +  Summary. Taken together, the data discussed so far suggest that the RC H mutations that impaired photosynthetic properties did so by reducing the amount of the RC complex in the cell membrane. These effects appear to stem from decreases in the amount of membrane-associated RC H , and override possible effects on the catalytic activity of low amounts of RCs that were assembled. The periplasmic and cytoplasmic domain substitutions greatly reduced the amount of membrane-associated R C H, whereas the transmembrane domain substitution functioned well for membrane targeting. A l l three domains of the RC H protein contain amino acid sequences that are required to obtain wild type levels of the RC complex in the cell membrane. Interestingly, the RC H-His 126/128 mutations did not affect photosynthetic growth of cells, although they clearly impaired purified RC catalysis in vitro. I explain this apparent paradox in the following paragraphs. The in vitro inhibitory effects of the simultaneous changes of RC H residues His126 and His-128 to Ala, were initially not seen when the catalytic activity of the purified R C was assayed in the standard Tris-HCI buffer. Subsequently, it was realized that the amino group of Tris (Fig 4.2) could act as a proton donor (because it would be partially protonated at the pH = 8.5 of the k B and A^assays), hence rescuing the effects of the A  His changes, on RC proton uptake. However follow-up experiments in the absence of Tris, addition of a variety of soluble proton donors (His analogues), and pH titrations revealed the function of His 126/128 as proton donors to a pathway leading to Q B (52). I do not present a detailed physical chemistry argument in support of these collaborative experiments, as frankly they are beyond my microbiological and molecular biological expertise. Instead, the interested reader is directed to the publications (2, 51, 52, 56).  93  Figure 4.2 Chemical structure of TRIS(hydroxymethyl) aminomethane. The N H moiety is in equilibrium between NH <-» N H states, which probably provides most of the buffering properties of this molecule (pKa =8.06) 2  +  2  3  The in vitro results on the purified R C indicate that the photosynthetic growth (Fig. 3.11) of the APUHAAPUC (p2xHis) strain may be explained by the vast variety of potential proton donors to the R C in vivo (e.g.; amino acids, and other cytoplasmic molecules), which could rescue the effects of the His-126 / His-128 -> Ala mutations. This could be categorized as an example of how a mutation may not result in a cellular phenotype, yet provide valuable information about a catalytic mechanism by careful in vitro analysis.  4.2 The role of the R C H protein in R. sphaeroides R C complex assembly The pioneering experiments of Kaplan and colleagues on R C complex assembly in R. sphaeroides, which included pulse-chase experiments on R C H and R C M , revealed a dynamic interplay between synthesis, membrane insertion and proteolytic degradation of these two proteins (64, 68). Their data led them to conclude that the R C H protein has a "chaperone-like" function in R C assembly, that the amount and stability of RC M in the membrane are dependent on the presence of RC H , and that R C H-stabilized RC M in the  94 membrane leads to "productive association" of RC M with R C L , resulting in R C assembly. However, the RC H mutation in the strain used by these investigators (in contrast to the chromosomal RC H mutations used in this thesis) appears to have a polar effect on the expression of genes located 3' of the RC H gene and deletes part of the 5' lhaA gene (3, 12, 64, 68, 72). These genes have been implicated as RC assembly factors, although their exact functions are unknown, which may account for the reduced amount and rapid degradation of RC M in the Varga et al. experiments (68). The RC H protein domain exchanges, and site-directed mutagenesis studies in this thesis indicate that the RC M protein and not R C H is the primary component in the process of RC complex assembly. Combinations of RC H , RC M and RC L gene deletions were constructed to investigate the possibility of stabilizing interactions between these proteins that affect their accumulation within the cell membrane and cytoplasm (see Materials and Methods section 2.2.3 for a description of mutants). As shown in Fig. 3.18A, all three R C proteins were expressed and inserted into the membrane of the control strain ARCLH(pESHPUF), consistent with the photosynthetic growth and the R C complex peaks in absorption spectra (Figs. 3.16 and 3.17). The western blot data (Fig. 3.18) indicate that the absence of either RC M or R C L results in the disappearance of RC H from the membrane fraction, whereas RC M insertion into and steady state levels in the membrane are independent of RC H or RC L . The amount of the RC L protein in the membrane fraction was reduced when RC H was absent, and undetectable when RC M was absent. It appears that R C M and RC L interact in the membrane in the absence of RC H , and so the western blot results are consistent with previous publications that described extremely  95 low amounts of RC catalytic activity in membranes of RC H" R. sphaeroides and R. rubrum cells (13, 64). Although it was previously shown that R C H and R C M proteins in the membrane are degraded if they are incapable of forming the R C holocomplex (68), the data in this thesis indicate that the rate of RC M synthesis and membrane insertion far exceeds the rate of degradation in the absence of RC H , because the steady-state amount of RC M in the membrane fraction is relatively constant irrespective of the presence of R C H . I also discovered that the amount of RC M in the membrane is independent of R C L (Fig. 3.18A). Thus R C M differs from R C H and RC L , which are absent or present at a low level when either of the other two R C proteins are not produced. These differences are attributed to degradation because the R C H gene transcription unit was identical in the plasmids pESHPUFM', pESHPUFL" and pESHPUF, and because the deletion of RC M gene sequences in pESHPUFM" is located 3' (transcriptionally downstream) of the R C L gene. The translationally in-frame deletion of the R C M gene in pESHPUFM" should not destabilize the 5' R C L mRNA segment because deletions of R C M gene sequences in the closely related species Rhodobacter capsulatus did not decrease the stability of the R C L mRNA segment (33). The data in Fig. 3.18B indicate that there is a maximal accumulation of the extremely hydrophobic R C M and R C L proteins (but not the less hydrophobic R C H) in the cytoplasm when all three R C proteins are present in the membrane, although minute amounts of R C M and R C L proteins were detected in the soluble fraction of the R C H" strain (compare Figs. 3.18A and 3.18B). These surprising results indicate that cells sense the presence of RC proteins in the membrane and somehow stabilize the hydrophobic R C  96 M and R C L proteins in a soluble form, the amounts of which roughly correlate to the amount of membrane-associated RC L. That is, the soluble forms of R C M and RC L were greatest when all of RC H, RC M and RC L were present, soluble RC L was not detected in the R C M" strain, but small amounts of RC M and RC L were detected in the R C H" strain. I use these collective results to propose the following model (Fig. 4.3) of the cellular accumulation of the three R C proteins for assembly of the RC complex in R. sphaeroides. 1) The synthesis of RC proteins increases in response to reductions in oxygen availability (48) with insertion of the three RC proteins into the membrane, followed by rapid degradation of RC H and RC L (but not R C M) if one or more of the other RC proteins is absent (Fig. 4.3 A). 2) RC M and RC L proteins interact (Fig. 4.3B), although RC L is especially susceptible to degradation in the absence of RC H . 3) The R C H protein interacts with the RC M / L heterodimer, in part through the R C H transmembrane domain and RC M residues, and protects RC L from proteolysis (Fig 4.3C). 4) The formation of a relatively stable RC H / M / L assemblage results in an increase in the levels of soluble RC M and R C L that may increase the rate of RC complex assembly (Fig. 4.3C). Perhaps the presence of RC L in the membrane is sensed by one or more phototrophic bacterial proteins that have been implicated as assembly factors (3, 12, 72) to result in this unusual accumulation of the extremely hydrophobic R C M and RC L proteins in the soluble fraction of cells.  97  A  RC L  RC M  RC H  B  C  Accumulation of RC M and RC L in the cytoplasm  Figure 4.3 Model for R. sphaeroides RC assembly in the lipid bilayer of the cellular plasma membrane (ICM). A Membrane insertion and degradation of RC L, R C M and R C H proteins, when synthesized individually under semiaerobic conditions; upward arrows indicate insertion into the membrane, while downward arrows indicate degradation; dashed arrow indicates a slower rate of degradation for RC M protein. B The RC M and RC L proteins interact to form a heterodimer in the absence of RC H , while the R C L protein is especially susceptible to degradation. C When all three proteins are present the R C H protein interacts with the RC M / L heterodimer, yielding a holocomplex; the formation of this holocomplex results in the accumulation of RC M and RC L in the cytoplasm.  98  4.3 RC-LH1 (core complex) interactions Although the focus of this thesis is on the elucidation of RC H functions in the R C of R. sphaeroides, absorption spectra of a number of mutants revealed potential RC-LH1 (core complex) interactions. For example, APUHAAPUC cells containing a mutant RC H protein with a stretch of seven deleted periplasmic amino acids (encoded in plasmid pEAPeri), exhibited significantly lowered levels of LH1 (Fig. 3.9), whereas the use of a hybrid R C H protein with a substituted cytoplasmic domain (encoded in plasmid pEVCSP) increased LH1 levels (Fig. 3.4). Also note differences in the amplitude of the. LH1 absorption peak in Fig 3.17. As discussed above, these mutations also affected the amounts of individual R C proteins in the membrane, and the R C holocomplex. Thus, the observed effects on LH1 indicate that the presence of the R C complex or mutations in R C proteins affect the amounts of the LH1 complex. Previous work on several purple bacterial species indicated a relationship between the amounts of the R C and LH1 complexes (3, 13, 64, 72). It would be very interesting to use the mutants described in this thesis to carefully evaluate the nature of LH1 and R C interactions.  4.4 Future Directions The data presented in this thesis are generally compatible with the conclusions drawn. Although many minor questions arise, there are several outstanding questions that warrant further investigation. For example: 1)  It is not proven whether the steady state amounts of RC H, R C M , and R C L proteins (as indicated by the western blots) really are the result of differential degradation, as opposed to changes in the rates of synthesis  and/or membrane insertion. This question could be addressed through the use of R C mutants in pulse-chase experiments, to evaluate the relative rates of protein synthesis, membrane insertion and turnover for the individual RC proteins. A number of RC mutations resulted in changes to the steady-state levels of the LH1 complex. Therefore, it would be interesting to determine the kinetics of LH1 complex assembly for these mutants in absorption spectroscopy experiments on intact cells. If LH1 a and p antisera were obtained, the membrane and soluble fractions could be evaluated in western blots, and in pulse-chase immunoprecipitation experiments, to study the individual LH1 proteins in comparison to the LH1 holocomplex. The speculated interplay between RC and LH1 proteins and/or cofactors, and putative assembly factors begs for additional research. Perhaps a combination of the steady-state measurements that are described in this thesis, as well as pulse-chase experiments, could be used to evaluate the effects of mutations in genes encoding cofactor biosynthesis enzymes and putative assembly factors. It would be interesting to use the R C M and R C L antibodies in immunoprecipitation experiments on the soluble forms of RC M and R C L. 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Bacteriol. 180:2801-2809.  109 APPENDIX 1: Amino acid alignment of R. sphaeroides and B. viridis R C H proteins  Cytoplasmic domain sequences  • Bv.puh.aa Rs.puh.aa  MYHGALAQHL DIAQLVWYAQ WLVIWTWLL YLRREDRREG gPUVEPLgLV MVGVTAFGNF DLASLAIYSF W-IFLAGLIY YLQTENMREG JYPIJENEDJG--  Bv.puh.aa Rs.puh.aa  TP§ANQ(3P-F  Bv.puh.aa Rs.puh.aa  gLQJPTGNgLV BAVGPASYAE gAEWDATVD § K A K W g L R V §TDFjSIAE(3D gHAfTCDgMK gGyGPAS.WVA fRDLPELDGH JGHNOKgMKA § A G | H V S A S K  14 9 14 6  Bv.puh.aa  V D g R G L P J V A AgGVEA^TjVjT  gLWVDRSgHY  F§YjL|LSVAG  SARgjAJDlgLG  199  Rs.puh.aa  -Ngi;GLPV RG  gl^VEilplQM  ASFLEVELKD  GS-gJRJDLgMQ  194  Bv.puh.aa Rs.puh.aa  FCD^KKDKIV P ' S I L S E Q F S NVgRLQgRDQ I fl]REEBKVS AyiYAGGL|LJg| MYK^QSNRVH j ^ N A L S g D L g s ] GIgTIK§PTE vjfELEEDRIC GMVAGGIiM|Yl  24 9 244  KL|PED§QVY  (  ELgYPKfglvS PfcgKp.KTFjlB  CgLEI^K^V  JPHG-^VJTVB;  raGR|lLfv|  RRRP|TlELK  f  Bv.puh.aa  TgEI?  Rs.puh.aa  AgKgKSWAA  AES LO MSAEYA  SSQ|DGFEGA  G P E S I D | P I A £35R|AVS|GF  50 47  99 96  258 260  1  APPENDIX 2: Absorption spectra of APUHAAPUC (LH2") intact cells containing plasmid pATSHR or pEVtrans (see Fig 3.7)  Time  Ohrs  18 hrs  25 hrs  70 hrs  640  760  880 nm  A P U H A A P U C (pATSHR)  640  760  880 nm  A P U H A A P U C (pEVtrans)  Ill  APPENDIX 3 : Publications arising from this thesis research 1. Adelroth, P., M . L. Paddock, A. Tehrani, J . T. Beatty, G. Feher, and M . Y . Okamura. 2001. Identification of the proton pathway in bacterial reaction centers: decrease of proton transfer rate by mutation of surface histidines at HI26 and HI28 and chemical rescue by imidazole identifies the initial proton donors. Biochemistry 40:14538-14546 2. Paddock, M . L., L. Sagle, A. Tehrani, J . T. Beatty, G. Feher, and M . Y. Okamura. 2003. Mechanism of proton transfer inhibition by Cd 2+ binding to bacterial reaction centers: Determination of the p K A of functionally important histidine residues. Biochemistry 42:9626-9632 3. Ali Tehrani, Roger C . Prince, and J . Thomas Beatty. 2003 Effects of Phtosynthetic Reaction Center H Protein Domain Mutations on Photosynthetic Properties and Reaction Center Assembly in Rhodobacter sphaeroides. Biochemistry 42:8919-8928 4. Ali Tehrani and J . Thomas Beatty. Effects of Precise Deletions in Rhodobacter sphaeroides Reaction Center Genes on Steady-State Levels of Reaction Center Proteins: A Revised Model for Reaction Center Assembly. Photosynth. Res. (in-press)  

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