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Studies on carbon monoxide and dioxygen binding to cytochrome P-450cam Rajapakse, Nimal 1984

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STUDIES ON CARBON MONOXIDE AND DIOXYGEN BINDING TO CYTOCHROME P-450cam.  By NIMAL RAJAPAKSE B.Sc,  University  of S r i Lanka,  1979  A THESIS SUBMITTED IN PARTIAL FULFILMENT THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE  FACULTY OF GRADUATE STUDIES (Department  We a c c e p t  this  the  THE  of C h e m i s t r y )  t h e s i s as conforming t o  required  standard.  UNIVERSITY OF BRITISH December ©  Nimal  COLUMBIA  1984  Rajapakse,1984  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may  be granted by  department or by h i s or her  the head of  representatives.  my  It is  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  N.  Department of  Chemistry  The U n i v e r s i t y of B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  2nd  RAJAPAKSE  January  1985.  Columbia  written  i i  ABSTRACT Interest decades  has  on  cytochrome  the  of  in  where  R-H  The  + 0  2  +  CO  and  0  2  to  understanding in  the  an on  various  properties  This  thesis  of  P-450cam.  b a c t e r i u m Pseudomonas  The  reduced  substrate-free  studied  using  0  calculated  (P-450)Fe(II)  binding were  not  +  to determine of  of  R-OH  are  a  +  dioxygen  successful.  to  wide  H 0 2  gas  molecules  important  not  monooxygenation  systems  bond.  that  can  such  only but  in  also  mimic  binding  studies  procedure  CO at  i s given  strain  P-450 to  carried  786  enzyme the  for  the  the  from  which  is isolated  CO  such the  ^  and  stoichiometrically  different  temperatures  thermodynamic  f o r the  out  growing  standard spectrophotometric procedure.  and  Attempts  widely  carbon-hydrogen  putida  enzyme  experimental data, were  A  of  these AS  gas  hydroxylating binding  a  model  system,  reaction,  small  enzymes  describes  the  purified.  of  two  P-450.  cytochrome  camphor  the  a s p e c t s of  on  soluble,  to  last  are  in oxygenation  >  binding  developing protein-free  catalytic  engage  unactivated  P-450  the  monooxygenase  2e~  +  during  hemoproteins  according 2H +  represents  Investigations as  intense  P-450  n a t u r e and  substrates R-H  very  heme-containing  P-450.  distributed variety  remained  parameters  was From AH  0  reaction, ^  (P-450)Fe(11)-C0  thermodynamic  parameters  substrate-bound  P-450  .  f o r the enzyme  iii  On  comparison  parameters  for  literature  values  the  myoglobin  and  substrate  molecule  the  of  substrate-bound  determined  substrate-free for  was  bonded  thereby system.  systems,  thermodynamic  system  substrate-bound  P-450 m o d e l  active-site  the  enzyme,  with  hemoglobin,  i t i s concluded  i n the  lowering  immediate the  CO  the  that  the  vicinity  of  affinity  to  the  iv TABLE OF  CONTENTS  Page  ABSTRACT  i i  TABLE OF  CONTENTS  iv  L I S T OF  TABLES  vii  L I S T OF  FIGURES  viii  L I S T OF  ABBREVIATIONS  X  ACKNOWLEDGEMENTS CHAPTER I  CHAPTER II  xii INTRODUCTION  1  References  6  LITERATURE REVIEW  7  11.1  Brief  History  11.2  N a t u r e and  Cytochrome  11.3  Structural  Considerations  11.4  The Mechanism o f C a t a l y s i s  20  11.4.1  Types  20  11.4.2  The  Binding  First  Binding  Second  Splitting  o f Oxygen-Oxygen  Oxidation  of S u b s t r a t e  Dissociation  11.5  Electronic  11.6  Interaction  of O x i d a t i o n  Catalytic  Carbon  of C y t o c h r o m e  P-450  .  P-450  15 17  Reactions  Cycle  24  of S u b s t r a t e  24  Reduction  25  of D i o x y g e n  26  Reduction  27 Bond  ...  27 28  of P r o d u c t  29  Spectroscopy  31  of Cytochrome  P-450 w i t h  M o n o x i d e and D i o x y g e n  References  8  34 43  V  CHAPTER  III  EXPERIMENTAL  PROCEDURES  49  111.1  Growth  I I I . 1.1  General  III.1.2  Media  III.1.2.1  Minimal  1 1 1 . 1.2. 2  L-Broth  I I I . 1 .2.3  500 mL  III.1.2.4  14 L F e r m e n t e r  56  I I I . 1.3  Growth  57  111.2  Isolation  of B a c t e r i a  50  Information  50 52  Agar  52 54  Shake  Flask  54  Procedure and P u r i f i c a t i o n  Cytochrome  of  P-450  63  Information  63  111.2.1  General  111.2.2  Materials  63  111.2.3  Buffer  Solutions  64  111.2.4  Column  Chromatography  68  111.2.5  Isolation  of Cytochrome  Cell-Free  Extract  Separation  Ammonium  111.2.6  Gel Filtration  Purification  Removal  111.2.7  Spectral  111.3  Determination at  Sulphate  69 69  of Cytochrome  P-450  ....  69  ...  70  Fractionation  Chromatography  of Cytochrome  o f Camphor  72  P-450  ..  Substrate  74  of E q u i l i b r i u m Temperatures  of Carbon  Substrate-Free  72 72  Analysis  Different  Reaction  P-450  f o r the  Monoxide  Cytochrome  Constant  with  P-450  ...  77  vi  111.3.1  General  111.3.2  Procedure  III.4  Determination for  Information  77 of E q u i l i b r i u m  the Reaction  Substrate-Bound  CHAPTER  77  of Dioxygen Cytochrome  Constant with  P-450  ..  80  I I I . 4.1  General  Information  I I I .4.2  Procedure  80  References  83  AND  80  IV  RESULTS  DISCUSSION  84  IV. 1  Growth  of Pseudomonas p u t i d a  Strain  786  85  IV.2  Properties  of Cytochrome  IV.2  Substrate-Bound  P-450cam  .  87  Cytochrome  P-450cam  87  IV.2.2  S u b s t r a t e - F r e e Cytochrome  IV.2.3  Purity  and S t a b i l i t y  of  P-450cam  91  Enzyme  Preparations IV.3  91  Determination at  Different  Reaction  of E q u i l i b r i u m Temperatures  of Carbon  Substrate-Free IV.3.1  Acquisition  IV.4  Comparison Values  f o r the  Monoxide  Cytochrome  t o Those  Hemoproteins  of  with  P-450cam  and Treatment of Measured A H  Constant  of Data 0  97 .  97  ....  106  and A S  0  Other  and Model  Systems  References  111  Appendices  113  vii  LIST  OF  TABLES  Table 11.1  Page Kinetic P-450  11.2  and R e l a t e d  Binding  Volumes for of  of IV.1  a Typical Cytochrome  IV.3  Binding  for Reversible  42  Solutions  Required  and  Purification 66  Required  Isolation  and  Purification  P-450 for  67 Substrate-Bound  P-450cam  89  f o r Substrate-Free  P-450cam  The E q u i l i b r i u m C o n s t a n t CO  Cytochrome  P-450  Absorbance Data Cytochrome  to  38  Data  Isolation  Absorbance Data Cytochrome  IV.2  binding  Systems  Q u a n t i t i e s of Reagents for  37  a n d Mb  of B u f f e r  Cytochrome  Cytochrome  to Substrate-Bound  P-450  a Typical  to  Systems  E q u i l i b r i u m and K i n e t i c  Cytochrome  111.2  Binding  f o r CO  and R e l a t e d  Dioxygen  111.1  f o r CO  E q u i l i b r i u m Data P-450  11.3  Data  93 Values f o r  to Substrate-Free  Cytochrome  P-450cam IV.4  K^ , A H  0  102 and A S  0  Some H e m o p r o t e i n s  Values  f o r CO  and Models  of  Binding  Cytochrome  P-450 IV.5  Half-lives  to  1 05 of Autoxidation  Substrate-Bound  Cytochrome  Reaction P-450cam  of 110  viii  LIST  OF F I G U R E S  Figure  Page  I. 1  The S t r u c t u r e  II. 1  The F i r s t Monoxide  11.2  11.3  The Carbon  Monoxide  Cytochrome  P-450  Electron  Record  3  of the Carbon  D i f f e r e n c e Spectrum of P-450  8  Leading  9  Reactions  t o Camphor Induced  Cytochrome  Benzopyrene  Difference Spectra of  a n d P-420  Transfer  The Substrate for  11.5  Published  Cytochrome  System 11.4  o f t h e Heme U n i t  in Bacterial  Hydroxylation.  Spectral  . . . 12  Changes  P-450cam  13  and i t sC a r c i n o g e n i c  Metabolite  7,8-Diol-9, 10-epoxide 11.6  The C a t a l y t i c  11.7  Hypothetical Substrates  11.8  'Proposed  Cycle  Mechanism  11.10  Valence  OD  6 6 0  Growth 111.2  OD  . . . 23  P450  f o r P-450  25 Mediated  Reaction  29  of Myoglobin,  Hemoglobin  Peroxidase  6 6 0  Growth  35  Bond S t r u c t u r e s  Cytochrome 111.1  P-450.  f o r t h e Binding of  t o Cytochrome  Heme E n v i r o n m e n t s and  of Cytochrome  Scheme  Hydroxylat ion 11.9  16  o f t h e CO-Complex o f  P-450  39  a n d pH P r o f i l e s i n the F i r s t  Set of Shake-Flasks  a n d pH P r o f i l e s i n t h e Second  f o rthe Bacterial . . . . 58  f o rthe Bacterial Set of Shake-Flasks  ... 60  ix  111.3  OD  6 6 0  Growth  a n d pH  Profiles  The C e l l - t o n o m e t e r  111.5  The  IV.1  The A b s o r p t i o n  10 cm  Spectra  IV.4  Cytochrome  Substrate-Free  Cytochrome  The A b s o r p t i o n  Spectra  Substrate-Free  and  States  Solution  Spectral  Changes no.  of 88  States  of  P-450cam  92  of O x i d i z e d  States  of  Substrate-Bound  Spectra  P-450cam  81  P-450cam  of Various  94 o f P-420  in Substrate-Free  Species  Cytochrome 96  Observed  f o r the  5  IV.6  A Typical  Hill  Log/Log  IV.7  The  Hoff  Plot  van't  Bath)  P-450cam  The A b s o r p t i o n  Experiment  (Slush  of Various  Spectra  Formed  IV. 5  Cell  The A b s o r p t i o n  Cytochrome  61 75  Path-length  Substrate-Bound  IV.3  Bacterial  i n t h e 14 L F e r m e n t e r s  111.4  IV.2  f o r the  P  1 / 2  98 Plot  100 103  X  LIST The in  this  commonly  following thesis  A  ABBREVIATIONS  abbreviations  along with  i n chemical  A  OF  a n d common  names  are  used  the standard abbreviations  used  literature.  Absorbance Observed  0  an  Ace  at the beginning of  experiment  Observed an  absorbance  absorbance  a t the end of  experiment  Buffer  P  50 mM  phosphate  buffer,  pH  Buffer  P-100  50 mM  phosphate  buffer,  pH 7.4,  7.4 100 mM  KC1 Buffer  T  50 mM  Tris  buffer,  pH 7.4,  Buffer  T-50  50 mM  Tris  buffer,  pH 7.4, 50 mM  Buffer  T-100  50 mM  Tris  buffer,  pH 7.4,  100 mM  KC1  Buffer  T-600  50 mM  Tris  buffer,  pH 7.4, 6 0 0 mM  KC1  BME  B-mercaptoethanol  o  Degrees  cam  o r camphor  Centigrade  D-(+)-camphor  DNase 1  Deoxyribonuclease 1  DTT  Dithiothreitol  EXAFS  Extended  X-ray  absorption  fine-structure  spectroscopy ENDOR  Electron  nuclear  EPR  Electron  paramagnetic  ESR  Electron  spin  AH  Standard  e n t h a l p y change  0  KC1  double  resonance  resonance  resonance  xi  Hb  Hemoglobin  max  Maximum  Mb  Myoglobin  NADH  Reduced  nicotinamide  adenine  dinucleotide NADPH  Reduced  nicotinamide  dinucleotide  phosphate  NMR  Nuclear  P-450  Cytochrome  P-450cam  Camphor  P-450LM  Liver-microsomal  RNase  Ribonuclease  A  adenine  magnetic  resonance  P-450  hydroxylating  P-450  P-450  A  RR  Resonance  Raman  SDS  Sodium  Ago  Standard entropy change  dodecyl  sulphate  xii  ACKNOWLEDGEMENTS I  wish  to  express  D.H.Dolphin  and  encouragement  through  thanks Lalith my  are also Talagala  deepest  gratitude  B.R.James out  for their  for  the  due t o M e s s r s .  appreciation  understanding.  my  help.  t o my  their  course Simon  of  for  Professors  guidance this  Albon,  Finally,  family  to  work.  and My  Gary  Hewitt and  I wish  to express  their  love  and  CHAPTER  2  I.  Introduction The  and  hemoproteins  activation  organisms.  hemoglobin,  the  terminal  formed  catalase  450)  is  last  the  its  The with an  to  (-S~  role  P-450 m o l e c u l e  ligand  hemoproteins dioxygen unactivated oxygen  generally  C-H  being  hydrogen  undertaken of  cytochrome  of  t h e most  of research  other  in  P-450  1 - 5  P-450  + 0  during  reflects,  i n part,  as a d r u g - m e t a b o l i z i n g  chemical  consists  moiety  carcinogenesis,  bond  of a  (Figure  from  single  protein  of  t o form  converted  + 2H +  and  to  1.1) v i a  the s p l i t t i n g one  oxygen  an a l c o h o l , a molecule  2e"  >  ROH  + H 0 2  chain  , joined a  a cysteine residue.  catalyze  insertion  +  2  (P-  unusual and  of atom  to  thiolate The  P-450  molecular into  an  t h e o t h e r atom  of  of water  (Reaction  1.1). RH  by  conducted  o f a p p r o x i m a t e l y 45,000  derived  and  oxidase.  of  number  in  specificity.  protoporphyrin  )  one  of cytochrome  substrate  a  i t s discovery  a m o l e c u l a r weight  iron  be level  since  i t s central  broad  while  t o water c  activation are  living  i s transported  by c y t o c h r o m e  hemoproteins,  intense  25 y e a r s  most  and reduced  systems  storage  a s mono- a n d d i o x y g e n a s e s .  a l l these  The  for  dioxygen  further  peroxidase  importance  enzyme,  and  considered  intriguing.  vital  by m y o g l o b i n  function  Amongst  transportation,  i n mammals,  in biological  and  hemoproteins  are  of r e s p i r a t i o n  decomposition  peroxide  the  stored  step  mediate  dioxygen  F o r example,  by  Also,  of  which  (1.1)  H  C H , C H  2  C O O H  ca C H  2C00H  The S t r u c t u r e of the Heme U n i t .  4  Therefore,  cytochrome  monooxygenases, incorporate  one  substrate.  The  hydroxylation high  stereo-  when  which atom  of  to  hydroxylation  bound  using  in  the  the  the  that  isolation  discovery  Peudomonas  of  pure  soluble  camphor  used  a  Reactions ground  state  be  overcome  in  turn  of  are by  react  organic  restricted  formation with  the  of  of and  form  P-450 on  model  into  in  system,  for  by  in  spin  in  the  6  P-450,  systems  resulted However,  7  bacterial  the  led  strain  to  present  P-450cam,  the study,  has  been  P-450.  with  dioxygen  conservation.  substrate.  same  membrane-  enzyme.  metal-dioxygen  organic  the  the  the  mammalian  compounds  out  cytochrome  D-( + ) - c a m p h o r  Therefore,  and  the  reagents.  of  with  remarkable,  if  purify  a  this  conditions  i n mammalian  monooxygenase  biological  fact  as that  out  carried  chemical  'inactive'  enzyme.  oxygen  result, be  solubilize  grown  in  history  soluble  enzymes  carry  reaction  to  enzymatic,  an  of  is  that  were  i s present of  to  known  physiological conditions  forcing  to  of  molecular  P-450  under  brief  put ida  isolation  as  non  class  from  products  attempts  enzyme  of  reaction  Throughout numerous  oxygen  the  collectively  a  regiospecificity  different  laboratory  of  reaction and  are  are  ability  compared  number  P-450's  8  in  the  This  may  complexes However,  the  which same  5  metal  complexes  also  react  hydroperoxides  to  Since  hydroperoxides  alkyl  generate  hydrocarbon  mixtures,  tend  mask  to  Therefore systems  preferably study  any  0  of cytochrome  only  in  biological  P-450 our  systems  that  capability  of these  enzymes;  industrial  potential.  the the  remain  such  site-substrate  aspects  was  the  some  to  in  Chapter  details  other  remarkable protein-free  0  activation  2  an  questions  unlimited  of of  known  the a c t i v e - s i t e In some  begins  with  the  of  present  insight CO  into and  an e x t e n s i v e  introduces various  by P-450.  experimental  Chapter  procedures.  experiments  are  hemoproteins  and model  c o n c l u s i o n s a r e drawn.  the  concerning  by s t u d y i n g t h e  I I , that  mediated  about  questions  to gain  interactions  IV, the r e s u l t s  comparison  not  bear  challenging.  focused  of monooxygenation  describes Chapter  survey  importance  the  function,  b i n d i n g t o P-450cam. The t h e s i s  literature  The  this  systems  unanswered  equally  active  and  mimic  oxidations,  conditions.  developing  of the substrates with  the attention  2  of  catalytic  9  catalytic  study,  0  selective  of great  most  activation'.  to develop  reaction  in  would  t o major  interactions enzyme  also  in  8  interactions  'oxygen  knowledge  catalytic  o f P-450  mild  i s thus  but  mechanism  via  alkyl  radicals.  ubiquitous  of promoting  under  2  widening  addition  are  importance  system  In  chain-initiating  reaction  are capable using  with  the metal-hydroperoxide  i t i s o f immense  that  directly  discussed  III In in  systems,  6  REFERENCES 1.  D.Y.Cooper,0.Rosenthal,R.Snyder,C.Wilner,Eds.,Cytochrome P-450 a n d b S t r u c t u r e , F u n c t i o n and Interaction, P l e n u m , New Y o r k , 1974. 5  2.  D.W.Cooper,H.A.Salhanick,Eds., Multienzyme Systems i n E n d o c h r i n o l o g y : P r o g r e s s i n P u r i f i c a t i o n and Methods of I n v e s t i g a t i o n , New Y o r k A c a d . S c i . , New Y o r k , 1973.  3.  T.E.King,H.S.Mason,M.Morrison,Eds.,Oxidases and R e l a t e d Redox S y s t e m s , U n i v e r s i t y P a r k P r e s s , B a l t i m o r e , Maryland, 1973.  4.  R . W . E s t a b r o o k , J . R . G i l l e t t e , K.C.Leibman,Eds.,Microsomes and Drug O x i d a t i o n s , W i l l i a m s and W i l k i n s , Baltimore, Maryland, 1972.  5.  M.J.Coon,R.E.White,Metal Ion A c t i v a t i o n of Dioxygen,(Ed. T . G . S p i r o ) , W i l e y I n t e r s c i e n c e , New Y o r k , 1980, p p 7 3 123.  6.  J.H.Groves,W.Kroper,T.E.Nemo, 7, 169 ( 1 9 8 0 ) .  7.  B . W . G r i f f i n , J . A . P e t e r s o n , R . W . E s t a b r o o k , The P o r p h y r i n s , ( E d . D . D o l p h i n ) , A c a d e m i c P r e s s , New Y o r k , V I I I , 1979, p.335.  8.  R.A.Sheldon,J.K.Kochi,Metal O r g a n i c Compounds, A c a d e m i c  9.  B.R.James,The P o r p h y r i n s , P r e s s , New Y o r k ; V, 1 9 7 9 ,  R.S.Myers,  J.Mol.Catal.,  C a t a l y z e d O x i d a t i o n s of P r e s s , New Y o r k , 1 9 8 1 , Ch.8.  (Ed.D.Dolphin), p.205.  Academic  CHAPTER I I  LITERATURE  REVIEW  8  II.1  Brief  The a by  History  presence  peculiar  kinetics  binding  while  1  was  not  first  1  of t h i s  P-450  microsomes  until  first  noted  pigment  b .  This  5  novel  1958, a t w h i c h  the carbon  microsomal  was  with  the oxidation-reduction  cytochrome  published  reported  of a pigment  spectrum  studying  microsome-bound  observation Klingenberg  monoxide  i n 1955  of  spectrum  in rat liver  carbon  Williams  of Cytochrome  time  monoxide-difference  (Figure  II.1).  450  20 10 — 0 T -10 -20  1 1 1 1  *-i  400  Figure II.1  440  480 520 560 Wavelength (nro)  600  The f i r s t p u b l i s h e d r e c o r d o f t h e carbon monox i d e - d i f f e r e n c e spectrum o f cytochrome P-450. (from r e f . 1 ) .  Although suggested  the  chromophore spectrum  the  Sato  2  the  showed  no  was  of  carbon  a  of  b-type  monoxide  heavy the  resemblance  metal  to  any  spectral  strongly  ion  electronic  hemoproteins.  conclusive  nature a new  of  pigment,  including  presented  hemoprotein pigment  presence  of  metalloprotein  binding  coloured  1962, Omura  evidence  for  the pigment  and c o n f i r m e d  cytochrome.  They  also  the  absorption  known  In  in  that  proposed  and the the a  9  'tentative' absorbs  name  a t 450  The  "P-450"  which  meant  "a  pigment  which  nm".  presence  of  a-  intense  Soret  bands  spectrum  of the  revealed  the hemoprotein  | 3 - bands,  and  in  reduced  the  carbon  pigment nature  in  addition  to  monoxide-difference  (Figure  o f t h e new  II.2),  clearly  pigment.  I t also  0.4 0.3 02  S 0.1 |  0.0  < -0.1 -0.2  400  450 500 550 Wavelength (nm)  600  650  F i g u r e I I . 2 The carbon m o n o x i d e - d i f f e r e n c e s p e c t r a o f cytochrome P-450 (A) and P-420 ( B ) . ( f r o m r e f . l ) .  was  observed  converted  that  i t  solubilized  "P-450", in  form,  focused  a l .  3  into  a  a prominent  with  detergents  spectrally peak  distinct  a t 420 nm  i nthe  spectrum.  the attention  photochemical et  with  the establishment  the study  became  of t h e pigment  quantitatively  CO-difference Since  treatment  of the hemoprotein  o f many  b i o c h e m i s t s who  of physicochemical properties on t h i s action  demonstrated  new  b-type  spectrum  technique,  f o r the f i r s t  time  were  of  cytochrome.  nature  of  engaged  hemoproteins  Utilizing  the  i n 1963, E s t a b r o o k the  participation  10  of  cytochrome  reaction  catalyzed  (Reaction  light  II.1).  reversal  enzyme  CO-difference addition,  function  with  that  nm  et  of  of  various Although  P-450  be  5  P-450  could  be  spectrum  able in  showed to  to  an the  intense reduced In  demonstrate  many  other  As  the  elucidated, by  the  mixed  hydroxylations  catalyzed  induced  the  microsomes.  physiological  was  for  monooxygenase  identical  monooxygenation.  enzyme  drugs. '  drugs  microsomes  active  including  the  cytochrome  this  action  liver  a l . " were  reactions  to  was  the  cytochrome  confirming P-450  that  C-21-hydroxylation cortex  microsomes  of  dealkylation  microsomes, cytochrome  these 450  oxidase  oxidative  adrenal  CO-inhibited  spectrum  of  steroid  photochemical  Estabrook  participation function  at  a  the  the in  peak  in  by  The  of  present  absorption  shown  P-450  by  and liver  function  of  nature  and  i t was  treating  also  animals  6  cytochrome  P-450  was  initially  thought  to  be  11  present of  a  only  five-year  microsomes, of  l i f e .  nature and  In  8  japonicum hence  not  could  crystallized.  1  material  2  class  the  next  challenge  very  researchers components  of  by  the  of  was  the were  the  the  look  with  for a  more  P-450-containing  membrane  and  1 1  provided  the  physicochemical  physiological the The  P-450  mechanism difficulty  system,  form  system by  function,  of  ionic  fragment  of  caused  a  The  cortex  treatment and  was  P-450.  adrenal  of in  which  membranes,  soluble  separated  Pseudomonas  8  the  11 -/3-hydroxylase finally  and  P-450.  microsomal  hydroxylating  has  determine  cytochrome liver  enzyme  and  conventional  homogeniety,  chemical  of  particles  camphor  to  species. Rhizobium  bacterium  enzymes.  to  associated to  mitochondria a  to  the  tightly  purified  of  establishment  supply  solubilizing  source  bacterium  the  the  convincing  bacterial  cell  from  mammals,  systems.  membranous  afterwards  studying  this  the  in  r e a c t i o n s as  first  in a  in  a l l forms  developed  the  matter  distributed  metabolic  in  isolated  This  is  highly  P-450  to  in almost  P-450  purified  Since  electron  into  was  for  p r o p e r t i e s - of  the  bound  and  1 0  of  partially  P-450  present  in a  7  discovery  monooxygenase  found  Shortly  9  cytochrome putida  be  of  tissue,  original  presented  9  presence  P-450  was  procedures.  component  the  cytochrome  be  b a c t e r i a to  Appleby  for  the  in d i v e r s i f i e d  activating  evidence  to  animal  cytochrome  primitive  1967,  of  from  shown  fact,  participates  In  best  period  i t was  from  oxygen  The  i n microsomes  1 3  - " 1  soluble  12  NADH-P-450  reductase  chromatography  into  flavoprotein. reconstitute and  the  of  was l a t e r  iron-sulfur  these  P-450.  In  1968,  system  protein,  (Figure  I I .3) .  NAD  REDUCTASE  1  from  F A D  +  and  required to the  was f o u n d  system,  t o be t h e  flavoprotein of  the  and  to  camphor  separated and i d e n t i f i e d  flavoprotein  a  a s an  cytochrome  P-450  5  >red  REDUCTASE (FAD)  NADH +H  of  NADPH-linked  PUTIDAREDOXIN (Fe )  P-450cam(SH) (Fe )  0  PUTIDAREDOXIN (Fe2 )  P-450cam(SH) (Fe )  SOH +H 0  2  2 +  3 +  C  were  constituents  were a  protein  s e p a r a t e d by  protein  components  iron-sulfur  of e l e c t r o n s  hydroxylating  +  which  the 11-/^-hydroxylase a c t i v i t y  transferring  iron-sulfur  an  A l l of  role  cytochrome  system,  2  3 +  +  Figure II.3 Electron transfer reactions i n b a c t e r i a l system l e a d i n g t o camphor (SH) h y d r o x y l a t i o n .  A  major  reaction  cycle  Narasimhulu binding cortex  breakthrough  et  of a l .  spectrum"  cytochrome 1  6  ,  microsomes.  microsomes,  the understanding  in  P-450  1965,  When was  the  added  the Soret  peak  of the  occurred  observed  of the C-21-hydroxylase  hydroxyprogesterone, cortex  towards  when  the "substrate  system  of adrenal  substrate,  to a suspension was o b s e r v e d  17-  of adrenal  to shift  to  13  a  shorter  original  wavelength, position  and  upon  substrate-induced  the  the  addition  spectral  P-450  also  the association  constant  t o b e ( 0 . 4 7 ± 0 . 07)  10 W  calculated  Pseudomonas  6  putida  to the  NADPH.  for  the was  II.4).  The  soluble observed  f o r camphor  (Figure  1  returned  of  change  cytochrome and  from  spectrum  binding 1  1 7  was  8  0 5  s B *!  01  0 350  Figure  400  450  900 550 W A V E L E N G T H <i>o0  600  II.4 The substrate-induced  650  7C0  s p e c t r a l changes f o r  cytochrome P-450cam. F i n a l camphor c o n c e n t r a t i ons  ( M) were, 0 ( 0  6 ( The  association  ) ; and 20.5 (  reaction  between  changes  the  iron(III)  detected  from  EPR e x p e r i m e n t s ,  in  2  Thus,  0  remarkable  The wide  from  a s i g n i f i c a n t change  iron.  microsomal  single  enzyme  1 9  step  system  3  species.  low  using  this Also,  ) ; and  ) , ( f r o m r e f . 18).  P-450  and  camphor  to high  spin  form  in  turn  potential  the  became  0  A  and t h i s  in  fractions  of substrates  technique,  a  i n t h e redox  first  biocatalyst  variety  spectrum  the  ) ; 2 (  W  results  o f t h e heme  reaction  well  of  shown  the  photochemical  i t  unusual was  this  understood.  were  being  as  to react  with  action  behavior  observed  a  that  f o ra the  14  cytochrome liver  P-450  had  essentially  although However, until  from  the  microsomes the  substrates  the explanation  later  cytochrome  same  of t h i s  separation  P-450  and  (P-450LM) as  450LMs,  designated  according  SDS-gel Some  of  characterized terminal  shown  many  as  2 7  multiple and  residues  di f ferences.  found and  forms  to six  other  of  2 1  delayed  microsomal  rabbit  liver  electrophoretic forms  P-450LM  2  (isozymes)  to contain  spectrum,  was  that  and  different.  to decreasing  e l e c t r o p h o r e s i s a s P-450LM,, these  very  observation  purified  contained  cortex  CO-difference  purification  homogeniety 2 2 - 2 5  adrenal  were c h e m i c a l l y  P - 4 5 0 . I n 1 9 7 5 , i t was  microsomal  of  P-  mobility in and so have  different  significant  of  C-  on.  2 6  been a n d N-  structural  15  11.2  Nature  All  and  Cytochrome  human  chronically  exposed  chemicals, disease,  beings  to  and  environmental  and  to  drugs  to  an  P-450  most  other  numerous  used ever  pollutants.  in  treatment  In  sustain  life  order  role  designing  'deadly'  xenobiotics.  forms  of  almost  a l l forms  oxygenating Cytochrome fungi,  is  reptiles,  2  P-450s  found  basic the  this  f u n c t i o n of lypophylic  However,  situation  of  cases  have  in  other  is  been  by  of  no  reported  in  versatile  mammals, liver, skin, and  8  are  additives,  pesticides  system to  excreted means  and  that  the  i s to  convert  more  polar  from  simple,  i n which  2 8  birds,  the  food  compounds be  many  yeasts,  i t i s conceivable  can  1958,  cortex,  products,  context,  these  fish,  tissues,  a  of  In  3 5  adrenal  drugs,  turn  3 2  this  reactions.  in  in microsomes  several  xenobiotic  which  number  mammals.  P-450 m o n o o x y g e n a s e  metabolites the  insects,  3 1  lung,  petroleum In  -  and  3  metabolising  2 7  be  3 0  • *  3 3  placenta  carcinogens.  to  in  occur  highly  present  testis,  anaesthetics,  a  to  known  small-intestine,  of  shown  in  biochemical  kidney,  capable  been  man-made  relatively  excretion  discovery  of  played  activating  f u n c t i o n i n g as  are  and  has  in diversified  amphibians,  cytochrome  its  bacteria,  9  nature  and  have  life,  catalyst  plants,  9  P-450  of  P-450  Since  to  and  detoxification  cytochrome  prevention of  remarkable  of  or  are  dietary  number  surroundings,  ways  earth  increasing  hazardous  efficient  on  non-nutrient  potentially  in  animals  the  body.  since  xenobiotics  a are  16  converted  to  more  hazardous  halogenated  benzenes  which  to  and  bind  converted  cellular  polycyclic  carcinogens  are  metabolites.  Figure  are  II.5).  '  their  macromolecules  hydrocarbons  (see  to  3 6  For  example,  arene  oxides,  3 7  causing  cell  converted Hence,  3 8  damage,  to  true  i t appears  that  F i g u r e I I . 5 Benzopyrene (A) and i t s c a r c i n o g e n i c m e t a b o l i t e 7,8-diol-9,10-epoxide (B). nature  has  enzyme and  failed  systems  thus  chemicals The  to  wide  the  soluble consists bound  allowing the  systems  from  example  and  of  a  P-450;  in  the  membrane-bound hydrophobicity  to  wider  of  designing  substrate  these  specificity  of  cytochrome  mammals of  were  how  to  this  changed. system  more  soluble the  a  in  harmless  carcinogens.  monooxygenase  form  extent  biotransformation  distribution  surroundings  bacterial  certain  potential  interesting  when  a  by  causing  monooxygenase an  to  enzyme  represents  system  A l l components  developed  microsomes,  bacteria  exist  reductase  P-450-containing  in  a  and  a l l components  and  hence  become  incorporated  of  have into  the  'primitive'  mitochondrial  component  evolved  a  system  membrane-  are  tightly  acquired  enough  biomembranes.  17  11.3  Structural Considerat It  of  i s of  the  cytochrome  understand reduction In  immense  this  the so  ions  importance P-450  the  discussion  P-450,  attention  450cam  enzyme,  of  will  of  be  since  in  order  reactions  focused is  mainly the  on  fully  and  dioxygen  be  mimicked.  of  the  most  structure to  can  s t r u c t u r a l aspects  it  the  oxygenation  biological the  elucidate  enzymes  mechanisms  that  to  cytochrome bacterial  P-  studied  and  most  bound  to  the  understood. The  heme p r o s t h e t i c  polypeptide and  hence  chain the  cytochrome.  3 9  polypeptide  via  group  an  system  the  attention  is  classified  cytochrome  chain  and  of  the  of  abnormal  substrate  binding  a  spectral and  compelling  indicate  the  presence  (RS~)  low  sphere been  ligation  Miyake that  has  the spin  and  of  shown  in  2  were  substrate-free (S=l/2)  form.  as  of  a of  a  protein.  the  heme  group  in  late  1960's  characteristics  a  b-type single  molecule  of  Since  has  linkage  3  of 9  '"  attracted because  observed then,  been  0  in  enough  gathered  to  sulfur  atom  in  the  first  the  heme-iron,  and  the  iron  sulfur  to  be  a l l four  Galor"  molecule  evidence of  covalent  single  CO-complexation.  and  distance  per  non  i s composed  investigators  direct  coordination  P-450  environment  many  is  labile,  The  immediate  P-450  acid  protoporphyrinatoiron(111) The  in  states  able ferric  This  consistent  to  of show  enzyme  the  enzyme."  using is  observation  with  EPR  thiolate 1  In  1968,  experiments  predominantly was  similarly  in  found  18  in  alkyl  ferric  and  5  high  nm  in  the  the  enzyme  However,  measurement  that  split  (RR)  can  of  the  react i o n s .  4  9  ligation As  the  the  of donate  CO,0  in  and  2  linkage  Recent  direct  (EXAFS)*  and  5 0  1  heme-iron.  in  the  case  the  of  ligand  residue  has  problem  water  still  l i g a n d has  the  remains been  Soret or  0  has  spin  P-450 A the  after  implicated  at  CO,  The  during  during  the Raman  absorption  or  by  indicated  fine  thiolate  hemoproteins, to  oxygen  candidate.  the  nm.  8  Resonance  ferric  extensive from  365  density.*  is believed  best  when  spectra",  confirmed  nitrogen  below  coordinated  intact  X-ray  as  peak  of  sodium  enzyme,  by  2  360  case  concentration  extended  low  around  "hyper  from  other  proposed  reduced  of  models  absorptions  evidence  coordinated.  been  of  remained  s u b s t r a t e - f r e e cytochrome  sixth  band  the  excess  proton  spectroscopy  to  Also  electron  exchange  Fe-S  iron(II)-porphyrins  porphyrins  high  and  spectrum  use  bands,  iron  the  in the  second  or  Chang  observed  obscured  Soret  changes  spectroscopy  structure  the  of  gave  mimic  Soret  not  to  of  ligation.  intensity  due  to  simple  was  hemoglobin  studies  able  CO  which  CO,  of  binding  and  ferric  stoichiometrically  with  which  were  7  which  characteristic  ligands  of  model  using  low  reductant  occurrence  are  the  energy,  CO-complex,  complexed  •"  6  ion  CO-complex  dithionite nm.  *  P-450  mercaptide a  the  others  carbonylated  showed  The  The  3  having  380  complexes  myoglobin.* '**  Dolphin" of  mercaptide  5 0  have  a  based Although  investigations, a NMR  relaxation  19  rate  of  water  resonance  protons  (ENDOR)  The  amino  consists  of  412  residues,  210  104  are  are  eight  half are  (54  four  situated above  450cam  molecule  sheet,  21  group  would  binding  and  the agent.  molecule,  which  I.C.Gunsalus, 5  is  with  four  Of  polar  amino in  acids.  the  half,  a-helix,  random  coil  and  and  a l l of  that  surrounded The  three  16%  number  by  hydrophobic large any of  enough  amino the  acid  described  as  of  the a  P-  pleted  amino  acid  prosthetic hydrophobic, pocket  for  thus  substrate  group  shape  in  the  5 3  5 3  extremely  dimensional  investigation  of  them  chain.  0-  the  mainly  There  5 3  2  structure.  the  and  NH -terminal  conformation  46%  these  neutral,  the  formation  been  heme.  that  prohibit  i s under has  basic)  cavity  of  The  50  conceivable  a  also  are  calculated  polypeptide  acids.  site  oxidizing  t a i l " . "  i t is  have  98  wi^th a  the  of  38%  enzyme  would  double  P-450cam  of  nature  amino  formed  to  interior  the  the  including  COOH-terminal  predict  present,  non-aromatic  and  consists  on  of  close  the  /3-turns and  Based residues  46,820  residues  the  data  nuclear  cytochrome  residues  hydrophobic,  in  in  of  acid  acidic  cysteine  and  The  of  are  ionic  sequence  amino  weight  electron  studies.  5 2  acid  molecular  and  5 1  being  powerful of  P-450  laboratory  "donut  with  of a  20  11.4  The Mechanism  1 1 . 4 .1 T y p e s  of Oxidation  Although enzymes of  that  can  activate  reference  Reactions  interest  rather  cycle  in  cytochrome  C-H b o n d s ,  a  will  described  be  brief  systems,  i t c a n be a p p l i e d  that the  discussion. with  types  The  particular  although  t o other  P-450  the d i v e r s i t y  of transformations  deserves  t o C-H h y d r o x y l a t i n g  modifications  lies  inert  and the v a r i e t y  execute  mechanistic  as  t h e major  substrates  enzyme  of C a t a l y s i s  with  of  minor  reactions  well. Guengerich  reactions (1). at  and  MacDonald  of cytochrome Carbon  P-450  into  hydroxylation:  a methyl,  methylene  classify  5 5  the  oxidative  s i x categories.  The f o r m a t i o n  or methine  of an  alcohol  position.  Eg.  (2).  Heteroatom  release;  heteroatomic  portion  hydroxylated  adjacent  geminal loses  hydroxy  The o x i d a t i v e  of  a molecule.  to  the  heteroatom  the heteroatom  t o form  cleavage  The m o l e c u l e i s  heteroatom  intermediate a carbonyl  of the  and thus  the formed  compound.  Eg. CH CH CH CH Br 3  2  2  2  >  CH CH CH CHBr(OH) 3  2  2  CH CH CH CHO 3  2  2  >  21  (3) .  Heteroatom  heteroatomheteroatom  oxyqenat ion:  containing  Conversion  substrate  to  its  of  a  corresponding  oxide.  Eg. Me N  >  3  (4) . E p o x i d a t i o n : from  o l e f i n s and  Formation aromatic  Me N=0 3  of  an  oxirane  derivative  compounds.  Eg.  (5) .  Oxidative  group  with  carbonyl  group  concomitant  at  C-1  transfer:  1,2-Carbon  incorporation  of  shift  oxygen  of as  a a  position.  Eg. C1 C=CHC1  >  2  (6) . O l e f i n i c heme  of  enzyme  The one  dioxygen, other  is  cytochrome  destruction; P-450  by  an  Inactivation enzyme  of  product  the  or  an  intermediate.  common  atom  suicide  CCI3CHO  of  feature oxygen,  i s being reduced  in  from  inserted to  a  a l l of the  into  water  these  the  reactions  reductive substrate  molecule.  After  is  that  scission  of  while  the  consideration  22  of  the observed  oxygen  experiments,the  hydroxylation, the  reductase  the  various  II.6).  stoichiometry,  5 6  and , a  t h e number scheme  steps  the  results  of  regioselectivity of  has been  electrons constructed  of the oxygenase  reaction  labelledof  required to  the from  illustrate  cycle  (Figure  Figure 11.6  The P-450 c a t a l y t i c  cycle.  24  II.4.2  The  As  Catalytic  i n any  reaction  This  iron(II)  bond,  that  induces  oxygen  as  the  by  the  of  an  dioxygen  and  a  inserted  step  of  of  i s returned to  A  of  forms  water.  Then  carbon-hydrogen  which the  to  second  dioxygen,  a  product,  the  iron(III)  molecule into  in  enzyme-substrate  binding.  splitting  alcohol  enzyme  first  reduction  for  is  the  the  formation  complex"  oxygen'  forming  released  the  allows  'active  catalysis,  i s followed  "active  the  is  which  reduction, the  enzymatic  cycle  complex.  Cycle  in  turn  original  is  ferric  state. II.4.2.1  B i n d i n g of  The binding state high  associated of  of  to  the  the  iron(III)  to  shift  5  ions  7  The has  a  second  equilibrium been  although  t h e r e may  substrate  and  responsible  the  of  for the  change the  order  are  the  rate  constant  some  enzyme.  the  to  hydrophobic  from has  causes  low  been  This  of  1  interactions  environment  1  8  from of  the  a  EPR  high  in the  1  The  at  main  in nature,  is  the  largely  entropy aqueous  active  1  potassium  between  bonding  increase  substrate  of  to  magnetic  6  hydrophobic  of  by  at  presence  type  spin  (S=l/2),  the  4.7x10 M ~ . be  the  3 . 7x 10 M" s"  of  5  to  the  observed  constant  be to  spin  proceeds  i n the  dipole  observed  lipophilic  enzyme  process  believed  be  c o n f o r m a t i o n due  measurement  binding  estimated  forces  into  by  The  1 8  bonding  transfer  This  1 9  and  1 9  susceptibility.  8°.  i n the  substrate  spectroscopy  with  change  the  spin,(S=5/2).  rate  Substrate  on phase  center.  5 8  25  Although  the  unknown, camphor  precise  inhibition to  bonding  be  in  site.  proceed  l o c a t i o n of  as  5  9  the  experiments  the  immediate  The  process  indicated  the  vicinity  is  bonding of  substrate  still site  the  of  oxygen  binding  could  II.7.  Figure II.7 Hypothetical substrates  show  of  in Figure  substrate  scheme  for  t o cytochrome  the  binding  of  P-450.  (from r e f . 58). The a  shift  free  most in  significant  redox  p o t e n t i a l of  cytochrome  of  -270  mV  is  -170  mV.  spin  (low  while  that  Since  2 0  change  spin) of  the  in  putidaredoxin,  change  upon  the has  substrate  heme a  iron.  E°  of  P-450  thereby  facilitates the  is  substrate-  potential  enzyme  putidaredoxin  enabling  The  reduction  substrate-bound  binding  (high  i s -240  (E°) spin)  mV,  the  reduction  successive  by  binding  of  transfer  of  dioxygen.  First  The an  next  electron  effort  Reduction step  to  the  devoted  questions  to  still  spectrophotometric  in  the  reaction  cytochrome. study  the  remain studies  cycle  Despite  nature  of  i s the the this  unanswered. 6 0  indicate this  considerable reaction,  some  Stopped-flow reduction  to  be  26  first  order  has  been  indicate  biphasic,  presented.  this  step  mechanism  of  iron(lll)  center  to  bind  protein, terminal II.4.2.3  the  enzyme  with  has  of  dioxygen  complex  temperatures  well  region  positions  thus  to  the  of  6 0  exact the  thought  the  from  the  in  I I . 6 . The  P-450  the  NH  2  1  at  1  indicates  that  the  presence  of  a  complex  of  as  the  4°  oxidized  adduct,  and will  substrate-free reaction  i s not  a  dioxygen,enzyme  fact  and  pH  7.4.  even  enzyme.  This  the  2  6 2  formed of  detail complex than This  3 0  in  dioxygen  camphor.  low  and  faster  complex.  The  process,  discussed in  times  t.o rate  structure  adduct and  from  at  enzyme-dioxygen 10  The  2 7 , 6 1  respect  decays  be  that  determined with  P-450cam  simple  the  second-order  enzyme-dioxygen  oxy-cytochrome  camphor  to  temperatures.  slowly  the  substrate-  ferrous-dioxygen  owing  enzyme,  M" s'  s  autoxidation  substrate-bound  the  is first-order  "autoxidation", dioxygen  the  low  reduced  P-450cam,  formed  give  of  Section  form  at  reduced  7.7x10  the  established,  stable  the  stability  ternary  Although  putidaredoxin i s  of  to  technique,  and  being  as  reaction  oxy-cytochrome  constant  the  calculated  putidaredoxin to  charged  412  behavior  Dioxygen  is fairly  dioxygen  undergoes  and  dioxygen,  been  stopped-flow  known  340  from  the  positively  association  formation  both  known,  this  constants  rate-limiting.  channeling  highly  B i n d i n g of  complex  the  rate  for  end.  complex, the  rationale  The  i s not  between  no  i s not  electron  to a  The bound  but  the but  27  Second  The steps  Reduction  ability (Figure  hydrogen  to  II.6)  peroxide  hydroxylated  reduced  enzyme-oxo  In  the  species  Unfortunately, either  the  because  of  The  model  the  of  the  to  c y c l e .  1 1 . 4 . 2. 5  S'plitting  The is  step,  cleaved  entire  6  ,  "active by  6  rate  2  6  5  produced do  6 5  system  6 3  of  6  not  possibly  6 5  agrees  camphor  well  with  hydroxylation,  determining  the  bond  between  is  step  and  in  the  Bond the  least  two  oxygen of  the  the  of  much  and  has  been  conversion  of  the  iron-peroxide  intermediate  molecule  coordinated unit,  of  to  the  water.  the The  heme-iron.  neglecting  subject  involves of  the  atoms  understood  scheme  heterolytic cleavage  two-atom  nature.  ji -peroxide. * -  ligand.  where  atom  this  in  2  structure  enzyme  Oxygen-Oxygen  of  on  for  electron  complexes,  reduction  the  two-electron  the  ferric  adding  7  oxygen"  a  that  the  The  the 2  the  by  external  Fe(III)-0 ~  thiolate  the  production stays  of  fourth  obtain  electrochemically,  power of  still  of  of  mechanistic  followed  or  heterolytically,  speculation. the  6  be  indicate  number be  catalytic  to  second  turn-over  appears  nature  and  cycle  and  Fe(III)-peroxo  6 4  absence  third  any  high-spin  oxidizing  rate  observed hence  be  synthetically  exibit  the  studies  the  enzyme,  without  complex  may  second, enzymatic  oxidized  that  model  the the  substrate  suggests  this  of  to  supply,  fact,  by-pass  0-0  complex  protonation, bond  with  remaining The  to  overall  contributions  the  oxygen charge of  the  28  porphyrin such  as  and t h i o l a t e ,  [Fe(lII )-0] , has been  this  "oxenoid"  with  single and  that  ligand by  type  oxygen  peracids  has been may  weakening  given  to the fact  atom.  iron  intermediates  If the  effects, reactions, elegant  ,C H I-0 6  5  similar  to  of  thiolate  by  an  electron-  explanation a  moiety  has  been  required  ligand  similar  peroxy-  hemoprotein  capable  undergo  i s the only  species  a l l point that  and  C-H  bond,  the  i t sreaction  with  interesting.  energy  regio-  examples,  of  porphyrins  of the peroxide  a t an u n a c t i v a t e d  bond  and  i s a necessary a  substrate  intermediate  active  energetically and  ferric  3 +  reaction cycles.  o f a C-H  hydrophobic  be  the occurrence  presence  i s not  r e a c t i o n , then  be t h e l o w e s t  would  P-450  are especially  strongly  the  which  iron-oxenoid  the cleavage  2  Substrate  an alkane  hydroxylation  would the  of  [ F e (V) - 0 " ]  system.  2  repulsion  thiolate  in their  cytochrome  hydroxylating  substrate  charge  or  designations  hydroxylations  the s p l i t t i n g  and p e r o x i d a s e ,  Oxidation  electrophi-lic  that  to  iodosylbenzene  B u t no d e f i n i t i v e  that  catalase  like  P-450,NADPH,0  7 0  ,  by u s i n g  to effect  9  suggested  for  Since  6  3 +  to demonstrate  donors  i t through  iron  >  facilitate  rich  of  7  leading -  intermediate atom  2  +3,  [ Fe ( I V ) - 0 ]  gathered  of the n a t i v e It  or  3 +  Evidence  6 8  becomes  site;  free-radical  could  any  ionic  species  Indeed,  isotope  stereo-selectivity  account  such  a mechanism.  for  in  that  unfavourable.  towards  step  the  exist  of 2 7  -  existence  in  these 7 1  '  7 2  of  Two an  .  29  uncharged Groves  of  et  substrate a l .  7  and  3  intermediate Dolphin  A  plausible  mechanism  the  substrate  radical  et of  a l .  have 7  been  and  hydroxylation  i s outlined in Figure  > R  Fe(llf )p+*  :cr,  by  4  formation  •«  discussed  H  <  II.8.  >  V  Z  7 1  H  A  •9 F«(lW)P  ROH + Fe(lll)P  :  F i g u r e I I . 8 Proposed mechanism f o r the f o r m a t i o n and h y d r o x y l a t i o n o f s u b s t r a t e (RH) r a d i c a l . The by  the  abstraction  active  species accepts  and the  of  iron-oxene a  Fe(lII)  hydrogen  complex  substrate  iron-bound  regenerates  a  atom  forms  from  an  Fe(IV)  free-radical,  hydroxy enzyme  radical ready  R*;  to  for  substrate  hydroxide  this  form the  RH  in  R-OH  next  turn and  reaction  cycle. D i s s o c i a t i o n The a  new  hydroxylated  cycle begins,  hydroxylations. may  be  coordinated  reduction even  The  i f the  of  the  iron  of  Product  product  since  diols  hydroxyl to  apparently  iron,  iron(III) i s reduced,  are  not  group but  center would  of  dissociates observed the  this more  in  product would  0  alkane alcohol  make  difficult  prohibit  before  2  and  the also,  binding.  30  Thermodynamically,  the  less  another  favourable  abundance spin  in  and the  reaction  cycle.  a  polar molecule  substrate molecule  surroundings  Fe(111)-substrate  another  b i n d i n g of  complex  will formed  be  that  preferred;  yields  the  will is  be in  the  high  way  for  31  11.5  Electronic  Spectroscopy  Electronic  absorption  extensively relation  to to  study  the  molecular  configuration  and  such  as  Mossbauer,  circular  structure,  (MCD),  nuclear  resonance  Raman  (RR)  unambiguously  the  spectroscopy.  Also,  weight  P-450  remarkably  the  with  enzyme  a  Upon  The  2 7  Soret  band  the  substrate-free  416  to  i n 500-600  intensity  nm  band  defined  the  nm  the  most  the  collapse  striking  low  on  molecular  forming  feature  spin  and  nm  is  band  is  new  form  a  formed. of  a  and  low On  bands. form  shift  of  distinct  energy,low reducing  potential to  408  nm  peak  at  542  nm.  when  /3  spin  a  moves  observed  state  hemeproteins,  with  new  are  spectral  d i s a p p e a r a n c e of  absence  a  sources  h e x a c o o r d i n a t e low  however,  Soret  0 bands  placed  ("resting")  well  391  643  ,ferric  and  0 ,  and  confirm  absorption  nm  such  2  and  electronic  known  ferric  i n the  or  a l l  spin  species  CO  to  and  low  region;  at  (NMR)  been  of  substrate-free as  from  electronic  binding,  band  has  (EPR),  circular  from  hemoproteins  to pentacoordinate high  Soret  bands  in  at  resonance  complexes.  is typical  substrate  changes  known  spectroscopic  used  reliance  in  electronic  resonance  obtained  used  system  magnetic  been  hemoproteins  similar  properties. of  heavy  iron-porphyrin  The  magnetic  results  other  Other  (CD),  have  been  P-450  paramagnetic  dichroism  has  geometry,  state.  electron  dichroism  with  cytochrome  oxidation  techniques  comparisons  spectroscopy  the  the  ligands and  the  a  However, reduced  32  enzyme peak  i s exposed  appears  at  hemoprotein. observed  at  peak  the  of  dioxygen a  low  446  The 540  nm  complex  peaks  about  species  at  the  552  gas  7r-7r*  These  spectra,  region, hyper  a'  380  region.  nm  The  exibit  studies" -" 5  in  spectrum,  are  But  spectral  bearing  CO  0  and  position  is  i n c l u d i n g the  using  iterative  extended  able  to  that  show  to  to  the  the  nm  and  (418  nm)  binding  as  a  the  and  well  bands  of  the  (split the  thiolate  Soret  by  reduced  to  Soret  by  show nm  a  of  number  Soret),  one  440-480  nm  species  the  model  an  axial  the  P-450  in  Hansen  et a l . "  calculations bands  UV  300-800  P-450  mimic  bands.  (IEH)  near  complexes  in  in  of  bands  characterized  exibited bands  of  considerations.  demonstrated  split  of  between  sets  Soret  other  Huckel  418  the  determine  three  required  split  as  2  the  Soret  presence  is  0 -complex  symmetry  pattern  As  complex  IV).  complexes  2  at  dioxygen  show  the  feature.  the  and  extra  two  a  spectrum  some p o r p h y r i n - m e t a l  strong  for  separation  visible, due  CO  band  efforts  Chapter  the  and  7  in  The  small  orbital  region  this  coordination  to  with  Soret  the  intense  intensity  a  studying  sharp,  the  same  nm)  a  wavelength  of  nm.  spectra  peaks  common  porphyrins  the  also  appear  transitions.  "hyper"  in  porphyrin  high  542  of  (408  (see  according  8  the  The  pressure  constant  bands  regions."  in  when  band  at  nm.  reduced  of  The  unusually  with  function  classified  an  energy  difficulties  Normal  atmosphere  low  created  equilibrium  CO  i s comprised  band of  a  nm,  reduced  energy  Soret  to  arise  from  B  were the  33  strong  interaction  thiolate  p-  porphyrin  TT-VT*  of  orbitals  charge to  transitions.  transfer  porphyrin  transitions  7r*-orbitals) with  (from the  34  11.6  Interaction  and  Cytochrome  observation  sodium  unusual (see  Soret  Section  inhibition  peak  binding and  to  Carbon  these  Monoxide  i t s model  to  hemoglobin  studied  (see  and  Figure  interesting the  stage  heme  II.9)  of  to  their the  molecule unit  are  and  Mb  note  is  normal  case  of  been  studies  differ The  Hb  of  same  in  many  important  and  Mb  and  and  P-450 6  1  -  CO  -  7 5  2  enzyme The  8 0  be  0  discussed  biomolecules  such  which  have  been  prosthetic  group  in  axial role.  8  not  undergo  prosthetic  groups  Mb,  do  ways in  an  biological  represented  the  difference  the  P-450  the  CO  will  (Mb),  cortex  of  on  of  understood.  contain  that  interacts,  spectrum  reported.  this  study  adrenal  studies  arv  P-450  of  a  reconstituted  such  play  state  and  well  they  must  many  in  cytochrome  of  action  myoglobin  and  monoxide  resulted  by  heme-containing  properties.  oxidation  In per  Hb  and  protohemin,  structure  reaction  have  from  other  (Hb),  Although P-450,  the  of  determined  native  compounds  and  thoroughly  was  carbon  biological function  findings,  reduced,  compared  the  of  microsomes  discovery  photochemical  gathered  in  with  addition  liver  the  . Also,  information  as  the  C-21-hydroxylase  via  to  to  enzyme  Since  3  led  I I . 1)  of  microsomes complex.  that  dithionite-treated  monooxygenase  as  P-450  Dioxygen The  to  of  their  ligation It  1  a  is  change in  any  heme  unit  functions. where  the  reaction  by  reaction  of  only CO  II.2,  one  with  the  reduced  Figure  I I . 9 Heme e n v i r o n m e n t s o f m y o g l o b i n and p e r o x i d a s e  (A), hemoglobin  ( C ) . (from r e f . 8 1 ) .  (B),  36  Fe(II)+CO where  Fe(II)  equilibrium dissociation association while  ^  ^ is  Fe(II)-CO,  the  constant, rate  the d i s s o c i a t i o n  kinetic  and  related  systems  respectively.  and k  Q  are  unit,  and k f f  n  < K  respectively.  data  a l l second  is  first  f o r CO  in  c  J I  «2) is  o  the  are association  D  reaction  given  on/'bf f  heme  i s an over  equilibrium  = 1 c o  reduced  constants,  reaction  K  The  binding  Tables  ligand  order  order.  II.1  82  and  process, .  83  The  t o P-450 a n d and  II.2,  37  Table II.1 Kinetic  Data  f o r CO  B i n d i n g t o Cytochrome  P-450  and R e l a t e d  Systems.  System  Method  PH  T°C  k  on  k  (M-'s- )  (s-  1  P-450cam  SF  7.4  4  5. 1 x 1 0  (cam.-free)  FP  7.4  5  2.7x10  s  1 5  4.7x10  6  25  8.4x10  6  s  P-450cam  SF  7.4  4  (cam.-bound)  SF  7.0  20  3.6x10  s  SF  7.0  24  2.2x10  s  FP  7.0  5  off 1  )  2.3  77  1 .2  82  3.8x10"  77 2.8  84  1 .7  75  8x1 0"  15  1.3x10  5  25  2.3x10  5  3.5x10"  Ref .  82  FP  7.3  12  Microsomal  FP  7.4  4  3.4x10  5  0.068  13  P-450  SF  7.5  4  4.5x10  s  0.63  88  FP  7.0  20  5x1 0  0.02  83  FP  7.0  25  3.8x10  23  1 . 1x10  Horse  P-450  Mb  Model  SF=stopped-flow,  FP  FP=flash  photolysis  85  5  86  s  s  18  87  38  Table II.2  Equilibrium Related  Data  for  CO  Binding  t o C y t o c h r o m e P-450 a n d  Systems.  System  PH  T°C  K  (M- ) 1  C 0  AH  As  0  (kcal /mol)  P-450cam  7.4  4  (cam.-free)  7.0  24  1 .7X10  P-450cam  7.4  4  2.6x10  5  (cam.-bound)  7.0  12  2.6x10  s  7.0  24  1. 3x10  23  1.1x10"  20  2.9x10  P-450  Model  Horse  Mb  7.0  2.2x10  0  (cal / m o l .deg)  77  6  7  Ref.  75  5  77 -12  -17  75 75  5  -16  -12.6  -35  87  -7.4  83  39  The  CO a n d 0  diamagnetic.  8 9  relationship that  carbon  of  The m e t a l - c a r b o n with  the  monoxide  molecular  orbitals  pair  the  of  complexes  2  P-450,  bond-order  C-0 s t r e t c h i n g  i s  involved  in  of the complex.  carbon  atom  Hb  i s  8 3  and  Mb  derived  frequency  a  both  are  from the indicates  and  u—type  The s p - h y b r i d i z e d  involved  lone  i n 0=C—> Fe  type  x bonding,  while  orbitals  of  the  t h e t w o pit iron.  CO c o m p l e x  orbitals  The P a u l i n g  are given N  .  overlap  valence  in Figure  N  Fe«—CsQ:  Protein  N  I  F i g u r e 11.10  Although transition  CO  i s thought  metals  ,  i t  to bind  the  chironomous  hemoglobin  CO  8 3  This  explanation towards Table the  access have  CO, c o m p a r e d  of  to  T h e camphor  heme m o i e t y the  7 7  =  0  in a linear  steric complex  observation  o f t h e lower  II.2).  Fe=C N N H  the  provides  affinity the  hindrance  a destabilizing However,  molecule effect  is  formed.  for  the substrate-free  f o r the P-450  system  (see  i n the v i c i n i t y to  t o t h e heme  restrict  unit,  at 24°, the equilibrium reported  the  basis  of the  but also to  o n t h e heme-CO c o m p l e x  system,  In  of substrate-bound  bound  in a  angle i s  substrate-free  molecule  .  to  Fe-C-0  a  i s considered not only CO  fashion  c a n be a t t a c h e d a t a n a n g l e  t o minimize  0  N  P a u l i n g v a l e n c e bond s t r u c t u r e s o f carbon monoxide complex o f cytochrome P-450.  heme p r o t e i n  145±15 .  s t r u c t u r e s of  -\/  \/ N  bond  t w o dir  11.10.  N  Protein  with  once  constant  by D o l p h i n e t  i t  value a l  7  5  ,  40  approaches  that  understand  better  to  P-450,  for  the  mentioned.  8 7  '  evaluation  of  200  reflects Mb, to  in  the  and  lowering  effect  (Table  to  kcal/mol  for  loss  would of  molecule.  Mb). be  But  that  Mb  (-12  Binding  unfavourable  the  and  entropy  of  Such  rationalized,  based  by  to  is  anion  atom, ligand.  thereby Since  9 1  this  could  constants.  value  a  from  of  terms  than  to  for  and  of  P-450 Mb;  in entropy c i s -  to  due  motion CO  system  P-450;  molecule  rotational  the  i s comparable  for  gas  binding  in  coordinated  calculated  0  This  probably  substrate-bound  AH  the  about  II.2).  thiolate,  in entropy  on  provided  imidazole  iron  kcal/mol  differences  the  binding  sixth  the  of  been of  Table  thiolate  than  the  has  P-450  ligand,  the  (AS=-17 c a l / m o l . d e g . )  cal/mol.deg.). been  indicate  binding  contributions  ligand  in binding  for  5  be  (see  parameters, 7  CO  to  temperatures.  proximal  a-donor  available  of  can  substrate-bound  f o r Mb  for  order  interpretation  entropy  The  In  studies  electron-rich  translational  unfavourable  have  an  difference  that  more  different  i n P-450.  weaker  II.2),  magnitude  centre  the  thermodynamic data  and  system.  s u b s t r a t e on  towards  affinity  is a  of  to  that  of  produce  f o r the  The  at CO  than  the  binding  affinity  the  imidazole  .limited  of  thiolate  heme may  account  values  weaker  difference  CO  enthalpy  binding  of  detailed  on  the  constant  times  effect  More  9 0  effect  The  the  substrate-bound  necessity  substrate  binding  the  in  -12.6 a  metal  to  the  the  gas  i s more (AS°=-7.4  contributions  trans-  effects  41  and as  steric the  e f f e c t s due  substrate.  model  system  entropy  7 5  -  contribution kinetic  and  dioxygen  binding  to  those  for  principal 0  2  CO  reason  complex  substrate-bound autoxidation minute, complex  of  faster.  The  to  this  the  (-35  II.3) of  well  protein  favourable,  data  available  P-450 a r e or  0  not  as  instability  enzyme  of  in  temperature occurs  to  free  but  the  date  for  extensive  binding  2  a  Mb.  the  pH  The  P-450case  of  7.0  the  half-life  of  i s at  to  the  and  with  autoxidation  substrate-free  as  cal/mol.deg.).  P-450,  room  protein  the  temperatures;  at  rate  for  i s more  i s the  ambient  6  H+  binding  of  times from  could  result due  to  electronic role.  for  +  2  mainly  site  so  of  the least  <  1  dioxygen 100  times  7 5  P-450Fe(lI)-0  least  term  0  cytochrome  (Reaction  the  presence  equilibrium  enzyme  while  9 2  AH  i s not  binding  at  the  kcal/mol)  The  as  The  7 7  (-16  to  a  >  dioxygen  weaker much  the  from  P-450Fe(III)  than  slower a  to  to  substrate  Mb  (Table  on-rate  in  geometrically  p r e s e n c e of t h e  e f f e c t s due  to  the  +  II.3).  the  (II.3)  2  bound  ligand  is  at  results  system;  restricted 7 5  P-450  This  P-450  substrate, sixth  H0  this  coordination although  may  also  the  play  a  42  Table I I . 3  Equilibrium  and  Substrate-Bound  System  P  Horse  Mb  Data  Cytochrome  1/2  (mmHg)  P-450  Kinetic  f o rReversible  P-450  Binding  7 5  Constant  (M- )  2.2x10  5  •52(20°C)  1.3x10  s  ^on 1  (0°C)  (20°C)  7.7x10  Binding to  k  off  (S-  1  )  ( 4 ° C ) 3.5 ( 4 ° C )  s  1 .4x 1 0  2  9 3  (M- s-M  1  2.5 ( 0 ° C )  and Mb.  0  7  ( 2 0 ° C ) 10. ( 2 0 ° C )  43  REFERENCES 1. M.Klingenberg, A r c h .Biochem. Biophys . ,  ,  376(1958).  2.  T.Omura,  3.  R.W.Estabrook, 741(1963).  4.  D.Y.Cooper, S . S . L e v i n , S.Narasimhulu, O . R o s e n t h a l , R.W.Estabrook, Science, 147, 400(1965).  5.  L.Ernster,  6.  S.Orrenius, J.L.E.Erricson, 25, 6 2 7 ( 1 9 6 3 ) .  7.  R . S a t o , T.Omura, O x i d a s e s and R e l a t e d Redox S y s t e m s . ( E d . T . E . K i n g , H.S.Mason, M . M o r r i s o n ) , V o l . 2 , J o h n W i l e y , New Y o r k , 1 9 6 5 , p.861.  8.  R . S a t o , T.Omura, C y t o c h r o m e Y o r k , 1978, p.24.  9.  C.A.Appleby,  10.  M.Katagiri, B..Gunguli, I.C.Gunsalus, 243, 3543(1968).  11.  K.Dus, M . K a t a g i r i , C.Yu, D . L . E r b e s , I . C . G u n s a l u s , B i o c h e m . Biophys.Res.Commun., 40, 1431(1970).  12.  C.Yu, I.C.Gunsalus, 1 4 2 3 ( 1970) .  13.  T.Omura, R . S a t o , D . Y . C o o p e r , O . R o s e n t h a l , E s t a b r o o k , F e d . 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Academic  CHAPTER I I I  EXPERIMENTAL  PROCEDURES  50  111,1  Growth  of  Bacteria  111.1 . 1 G e n e r a l The 29607) and  Information  bacteria derived  Wagner  cytochrome isolated strain  used  P-450. from  was  plates  from  was  1  Pseudomonas  PpG  throughout The  soil  kept  strain  putida  parent  by  alive  containing  1  (ATTC  PpG  studies  strain  PpG  bi-weekly  on  (ATTC Gunsalus  to  1 was  produce  originally  D-(+)-camphor.  transfer  D-(+)-camphor  786  1 7 4 5 3 ) by  these  enrichment by  strain  as  The  to minimal  carbon  and  agar energy  source. The  four-stage  production  of  was  with  used  procedure  expense short  of  period  much  of  of  the  the  carbon  for  Gunsalus  modifications. maximum  monooxygenase media  with  PpG  786  the  a  and  and  the  Wagner  This  components has  a  ability  of  at  the  relatively of  to  release  more  breakage  techniques.  liberates  about  cycle  cell  monoxide  The  four  1.  Minimal  typically  P-450 based  cell  content  on  the  suspension difference  stage agar  growth stage  as  as  total  freeze-thaw  about  by  tedious  soluble amount  determined  spectrum. procedure,  autolysis  one  enzyme of  from  1  growth  amounts  g e n e r a t i o n time  has  mass  hydroxylase proteins  extract,  whole  by  produce  liquid  strain  cytochrome  cell-free  to  growth  freeze-thaw  the  described  other  and  of The  circumvent  single  and  procedure  minor  designed  camphor  hour's.  as  several  P-450  three  to  bacteria  is  cytochrome  growth  A  half  in  the  enzyme  in  the  ferrous  51  2.  L-Broth  3.  500  4.  14  typically weight  L  Shake  i n about  carried  inoculated  in a  of  each  each  use,  i n the  the  shake  platform  (Labroferm;  12  L  aeration. of  growth  measuring Lomb  the  in  210  optical  the  bacteria  centrifuge 37,  000  of  mL  of  pH  rpm.  were  (Carl The  to  was  was  rpm  660  was  monitored  the  with  medium  collected  Padberg, bacteria  a  to  red  then  hot  before  medium  at  and in  used  was  made  up  L/min  rate  of  30°.  During L  using glass  The  rate  Bausch  the  by and  growth  of  fermenter  stages,  a  Accumet  Fisher  pH-electrode.  reached a  a  fermenters  U.S.A) were  15  the  L-broth  L  a  When  maximum  value,  continuous  flow  Schnell-Zentrifuge, were  were  transfer  using a  14  using  minimize  turbidometrically  nm  and  to  plates  14  with  determined  flask  were  incubated  N.J,  S p e c t r o n i c 20.  shake  fermenters  The  Three  maintained  at  wet  until  growth  300  the  agar  and  30°.  The  on  station  burner.  Scientific,  at  L  used  heated  Bunsen  density  of  loop  inoculated  equipped  density  14  Three  wire  based  time.  clean-air  growth.  model  pH-meter  the  a  were  optical  fluctuation  model  of  bacteria  500  bacteria  for  was  temperature  spectrometer  bacteria  the  stirred  of the  flow  and  flame  stage  The  except  Brunswick  and  of  elapsed  thermostated  New  final  of  plates  flasks  shaker  g  contamination.  time  between  to  hours  laminar  bacteria  the  500  inoculations out  stage  stage  about  96  possibility  in  flask  Fermenter  yielded  All  the  mL  stage  stored  Model  under  LE)  at  dry-ice  52  temperature  I l l . 1 .2  i n a Dewar  for  a l lfour  Gunsalus  and Wagner  freshly  distilled  under  Co.  than  200  possible  In plates  Minimal order  were  an  20 m i n 45  min  Solutions  were  store using  the b a c t e r i a the following  0.06  g  2  0.05  g  (NH ) SO„  0.5  g  0.68  g  7.5  g  NaCl MgSO«.7H 0 2  D-( + )-camphor agar  Water  sterilized,  L  complete as  early  minimal  agar  ingredients.  g  salts,  14  3.5-g  a  Bacto  volumes  while  alive,  1.0  a  of  used  at  (American  ensure  solutions  and  sterilized  autoclave  to  2  petri  were  autoclaving  KH PO«  The  chemicals  re-contamination.  prepared  2  using  according to  Agar  to  K HPO  grade  A l l solutions  sterilized  to avoid  prepared  AS-DTT616GE).  required  The  were  reagent  steam  required  sterilization.  111 . 1 . 2.1  water.  Model  mL  fermenters  as  batches.  stages  using  1  pressurized  Sterilizer less  g  Media  Media  121°  i n 500  540  camphor  cooled,  dishes obtained  and agar  combined from  were and  Canlab.  mL  dissolved poured  in  into  The m i n i m a l  water,  disposable  agar  dishes  53 were to  stored  inoculate  at  5°, three  with  the  of which  bacteria.  were  used  every  two  weeks  54  III.1.2.2  L-Broth  At  the  bacteria,  a  facilitate following  initial  very  rich  the  rapid  stage medium  flasks  large  called  and  mixed,  0.5  g  NaCl  0.5  g  Glucose  0.1  g  extract  500  this  stock  source  was  shake  flask  acid  as  stage  the  camphor  as  1  medium  and  L  of  the  divided  salts  to  pH  1 00  mL  7.0  by  The  five  50  adding  1M  was  salts)  the  and  prepared five  and  P-450 twice  energy  carbon  the  medium.  performed and  phosphate-ammonium  s u p p l i e d from  (100-X to  a  maintain  cytochrome  only  into  to  were  added  carbon  was  growth  used  solution  of  organism.  mL  NaOH  Flask  of  was  also  to  sterilization.  stage  production  brought  Shake  mineral  strong  the  mL  solution  Essential  was  the  used  sterilized.  Yeast  before  of  into  g  I l l . 1 .2.3.  buffer  the  1.0  solution  growth  was  2  Bactotriptone  This  At  of  poured  Water  dropwise  scale  L-broth  multiplication  i n g r e d i e n t s were  Erlenmeyer  of  500  mL  In  the  a  pH  carbon order  and to  the  , first  with  and  source.  energy  maximize 500  mL  glutamic  secondly  with  In  case  following  Erlenmeyer  level.  hundred-fold  content,  source  energy  using  a  desired  (PA)  each  ingredients flasks  and  55  sterilized.  PA  buffer  100-X  PA  salts  16 mL  Glutamate  2.9 g o r  Camphor  2.5 g  buffer  following  PA  1 L  and  100-X  salts  were  prepared  ingredients.  buffer: K HPO,  10.7 g  KH PO«  3.1 g  NH«C1  4.0 g  Water  1 L  2  2  100-X  salts: MgSO,.7H 0 2  19.5 g  MnSO,.H 0  5.0 g  FeSO«.7H 0  5.0 g  CaCl .2H 0  0.3 g  2  2  2  2  L-Ascorbic Water  acid  1.0 g 1 L  by  mixing  56  I I I . 1.2.4 The  14 L final  somewhat but with  Fermenter stage  similar  i t was  following  growth  medium  essential  camphor  of  chemicals  the  the  whole  were  carried  t o t h e 500 mL  t o keep  during  was  added  shake  medium  of  g  KH P0„ 2  36.3  g  NH,C1  46.8  g  120  mL  2  fl  100-X  salts  Bacto  yeast  extract  Antifoam Camphor Water  i n DMF  (3M)  3.0  g  1 .0  mL  30  mL  1 1 .6 L  in  a  stage,  saturated  growth.  fermenter.  128.2  K HPO  flask  always  period  i n each  out  The  57  111.1.3  Growth  Four mL  Procedure  of the five  Erlenmeyer  o f L - b r o t h medium,  shaken  At  The each  Each 3.2  mL  density  induction  initiated  one  were  of  the  every  continued  at OD  thereupon  until  flasks  6 6 0  )  hour  reached  of stock  camphor  (seeFigure  the  III.l).  flasks  with  source  was i n o c u l a t e d  inoculation  as a  time  each  medium  with  5 mL  reference.  PA b u f f e r  ,  as the carbon and a t 30° u n t i l t h e 0.3. A t t h i s system  solution 6 6  o  the of  time  a  n  was  (3M  in  d t h e pH flask  growth  and  camphor  was  three  hours  i t s late-logrithmic the second  as the only  10 mL  the culture  flasks  every  reaches  At t h i s  camphor with  once  the  inoculating  and the r e f e r e n c e  Addition  and  growth  by  The O D  t o monitor  camphor.  0.7, 1.0  6 6 0  flasks  while  seen.  of basal  shaken  a  noted  swirling  5-monooxygenase  i n order  of  i t was  be  served  glutamate were  camphor  inoculated  shake  this  a n d 10 mM  (OD  could  c o n c e n t r a t i o n o f 5 mM.  metabolism  phase  200 mL  by t h e a d d i t i o n  taken  the  contained  of  DMF) t o a f i n a l of  flask  a t 660 nm  On  shake  as  turbid,  was s t a r t e d  The f i f t h  source. A l l five  optical the  flasks  served  period  clear.  20  t h e b a c t e r i a and  turned  500 mL  containing  flask  of bacteria  of growth  o f 100-X s a l t s  energy  remained  s e t of four  culture.  of these  The f i f t h  inoculated  clouds  stage  of the f i r s t L-broth  were  each  with  o f t h e 10 h o u r  flask  flasks,  third  end  that  reference  inoculated  of  the  the flasks  the  inoculated  a t 3 0 ° f o r 10 h o u r s .  reference. that  were  flasks,  carbon  of this  was u s e d  s e t .of  and energy  solution. For only  from  the  58  59  three at  shake  30°.  and  flasks  The  flask  optical  inoculum,  in  for  they  that  heated  to  pipettes  as  the  to  that  but  red  14  flasks  were  were  allowed  aeration  used  (see  solid  respectively. preferred camphor However  The  tended ,  the  to  the  camphor  from  reached  14  under  which of  the  at  clog of  a  a  the solid  flasks. camphor  smaller  compared  Figure  III.2),  i t s late-logarithmic  phase  the  values  three  fermenters.  the  the  OD  of  6  6 0  (3M)  bacteria  of  continuous  as  well  or  since  flow  0.6  camphor  6.7  camphor  helped  and  reached  mL/h  growth  camphor  The  agitation  15  solution of  undisturbed  feeding with  i n DMF  rate  stages  Pasture  about  until time  was  of  continuous  camphor  of  late  L  or  after  of  I I I . 1.1) at  similar  reached  contents  (see  burner  disposable  2 mL  was  glutamate  neck  solution  Pre-sterilized aliquots  of  camphor  1 mL  source  of  an  the  of  energy  use  use  the  as  Bunsen  and  used  towards  a  presence  grow  was  of  for addition of  pH  used  around  flame  needle  for  possibility  i n the  solution  camphor  any  growth  Section  A  never  heated  the  use.  inoculate  to  was  shaking  aliquots  of  the to  on  The  growth  III.3),  started.  were  withdraw  density  hours),  Figure  to  the  withdraw  avoid  either  before  presence  When  undisturbed while  to  to measure  rate  optical  hours.  (see  used  carbon  in the  to  opened  used  only  used  rotating  hot  left  measurements  aliquots.  was  (about  was  by  initial  the  was  flasks  were  were  The  24  The  withdrawing  syringe  were  order  opening,  whenever  that  density  contamination. before  that  the  as g/h, was  solid  centrifuge. to  reduce  the  T  i  i  1  1  1  1  1  1  1  1  1  r  inoculum for the 14 L fermenters  Time (h) Figure III. 2  O.IXgg  n  and pH p r o f i l e s f o r the b a c t e r i a l  growth i n the second s e t of s h a k e - f l a s k s .  61 to in  I  i  I  1  r  i  1  1  r  1  to  to r-'  readjusted to 7.4 to tn to  to to'  '  '  I  3  to l _  L  J  L  harvest  r d  o O  —  CO  J  0.0  16.0  Figure III.3  32.0  48.0  I  I  64.0  L  J  80.0 Time  O.UggQ  and pH p r o f i l e s f o r the  growth i n the 14 L  fermenters.  I  96.0 Ch)  I  L  112.0  bacterial  62  o f DMF a d d e d  amount excess  DMF  of  centrifugation 1.2  collected.  and to of  again  enough  100-X  the  optical  the  procedure stored being  used  the O D g  reached  1.2  (about  to bring was  the 6  6  6.0  at  hydrogen t h e pH added  also  2  to 7.4.  in order  1.2.  The  total  about dry-ice  was  5.5  450-500  days.  (-78°)  t o i s o l a t e cytochrome  this  time  was  added  120  out again wet  weight  g. The t o t a l  The p a s t e  of  for at least P-450.  last  to replenish  carried  collected  had  c a r r i e d out  Another  was  reached  about  from  (K HPOi,)  phosphate value  The  hours was  below  growth.  the o p t i c a l  the c e n t r i f u g a t i o n drops  an  bacteria  until  24  that  reached  0  of  centrifugation  density  takes  150  about  continued  final  under  o u t when  was  time  likely  witlr  o f t h e medium  bacteria  is  growth  salts  The  It  interfere  and  potassium  t h e medium  media.  carried  a t which  T h e pH  medium.  was  The  harvest), again.  may  24 h o u r s )  (about  density  to the  the when of  growth  bacteria 10 d a y s  mL  was  before  63  I I I .2  Isolation  111.2.1  General  The  to  and  putida  Wagner  Basically,  cytochrome  the  debris  by  buffer  solution,  filtration  P-450  111.2.2  Materials  The  Co.;  grade  a  G-10  involves cell  camphor  filtration  and  and  ammonium  0-mercaptoethanol  G-100  from  sulphate (0ME),  by  a  from  Pharmacia  from  Once  from  bovine  pancrease,  isolated  from  bovine  pancrease,  other  of gel  isolated in  liquid was  a  exchange  liquid in  removed  the by  procedure.  as  follows.  Whatman Fine  Chemical  Chemicals;  Schwarz/Mann  deoxyribonuclease  isolated  cell in  from  frozen  a  were  52)  of  two-step  chromatographic  (DE  Gunsalus  anion  substrate  sources  cellulose  P-450  stored as  were  purification  done  kept  of  removal  procedure. be  III.1.3)  suspension  The  was  bacteria  modifications.  two-step  can  be  minor  cytochrome  P-450  The  gel  Diethylaminomethyl  Enzyme  by  can  chemicals  Sephadex  method  stirred  samples  (5°).  two-step  The  procedure.  or  another  Section  chromatography  (-196°)  refrigerator  P-450  of  (see  some  separating  cytochrome  column  purified,  nitrogen  Co.;  of  components  crude  cells  procedure  chromatography  isolated  and  with  centrifugation and  786 P-450.  used  isolation  monooxygenase  Cytochrome  autolized  strain  was  1  of  Informat ion  isolate  column  Purification  freeze-thaw  Pseudomonas used  and  ribonuclease-A dithiothreitol  Chemical 1  (DNase) (RNase)  (DTT)  and  64  t.r i s - ( h y d r o x y m e t h y l ) a m i n o m e t h a n e Chemical  Co.;  potassium  Chemical  Co..  I l l .2.3.  Buffer  All  by  column  with  of  heated  solutions  water,  flushing  argon.  copper  prepared  from  III.2.2.)  solution  followed  Four  buffer  different KC1  to  these  a  ionic  10 mM  solutions  are  stirred  stock  Prior 0ME  (4  The  was made  Chemical  followed  buffer  to  mM  50  of each) 0,  were  T,  Drierite  Section  distilled  a pH  5 0 , 100 a n d  T-50,  were  o f 7.4.  prepared  For s i m p l i f i e d  as  drying  (see with  a  Co.)  solutions  Base  by a d d i n g  by  Indicating  Trizma  L  through  with  600  mM  nomenclature  T-100,  T-600,  solutions  were  .  of  solutions  1 M K HPOi, 2  a pH v a l u e  100 mM  f o r about  passed  to use, a l l the buffer  50  was  followed  (BASF  and  buffer.  buffer  to obtain  aspiration  o f 6M H C l t o o b t a i n  specified  volumes  camphor  Scientific  glass-  dioxygen,  by d i l u t i n g  Tris.CI  with  solution.  Solid  1.0M  phosphate  appropriate  Sigma  from  was  pellets  pentoxide  strengths  mM  buffers  buffer)  argon  trace  solutions  50  The  mM  The  by a d d i t i o n  respectively. made  prepared  D r i e r i t e C o . ) . The T r i s . C I  water  from  American  by r e p e a t e d  catalyst  phosphorus  (Hammond  from  were  deaerated  t o 50° t o remove  through  chloride  Base)  Solutions  buffer  distilled  (Trizma  mM  6 hours  and  M  o f 7.4, when  KC1  t o produce  obtain  a  by  mixing  KH PO«  stock  2  diluted  buffer  to appropriate to  prepared 1  phosphate  with  added  were  to a  solution buffer  buffers saturated  50 (P-  P-100.  that (8  were mM)  65  solution. The typical  approximate preparation  bacteria  are  reagents  used  listed are  volumes  of  of  buffer  cytochrome  in  listed  Table  P-450  111. 1 .  in Table  III.2.  solutions  used  from  about  400  The  quantities  in a g  of of  66  Table III.1  Volumes  of  Isolation  and  Buffer  Solutions  Purification  Required  of Cytochrome  Step  Cell-Free Anion  Extract  Exchange  Chromatography  for  a  Typical  P-450.  Buffer  Volume  T  0.6  L  (DE52)  (a)  Equilibration  of  column  A  T  4.0  L  (b)  Equilibration  of  column  B  T-50  4.0  L  (c)  Separation  T-1 00  4.0  L  T-600  4.0  L  T-1 00  1 .0  L  1 .0  L  of  protein  components  Gel-  Filtration (a)  Column  (Sephadex A  (1OOmM  -  (b)  Column  G-100)  B  DTT)  P-1 00 (8  mM  Cam.)  67  Table  III.2  Quantities  of Reagents  Purification  Required  of Cytochrome  Reagent  base,  KH P0 , K HP0 ,  4  2  4  P-450.  1M  800  mL  1M  20  mL  1M  35  mL  KC1  250  (NH ) 4  2  NH 0H,  I s o l a t i o n and  Quantity  Trizma 2  for a Typical  S0  4  15M  g  100 g 2  mL  B ME  10  mL  DTT  0.2  g  4  DNase  1 .0  mg  RNase  1 .0  mg  68  111.2. 4 Column Since  Chromatography  the  cytochrome  success  P-450  of  isolation  very  much  exercised  i n the p r e p a r a t i o n  operation  of  columns  for  by  Chemistry  columns  were  100  mesh  in  peristaltic  gravity Broma,  The  samples apparatus membrane  The  enzyme  Ultrarac  were  Sephadex  samples  fraction  ( 1 0 , 000  with  of  columns  a  molecular  and  flow  the and  were were  an  100  were  collected and  Diaflow  weight  cut-off).  mesh  columns using  with the  a  constant  equilibrated  YM-10  DE-52  only  mode  Amicon  of  contained  DE-52  collector,  using  were  column  Instruments) to achieve  concentrated  fitted  ends  Both  columns  The  i n the m e c h a n i c a l shop  descending  Sephadex  care.  (Model Bio-Rex)  porous polyethylene  (Buchler  and  the manufacturer's  filtration  U.B.C. The  of  of  precision  chromatography  gel  inserts. the  pump  feed. 7000  constructed  ends  the  special  Laboratories  were  nylon  equilibrated  with  Sephadex  The  on  columns,  exchange  with  purification  chromatographic resins  for  fitted  inserts.  rates.  anion  Department,  nylon  flow  of  followed  Bio-Rad  columns  chromatography the  were  DE-52  manufactured the  depend  the chromatography  recommendations  and  under a  LKB  enzyme  ultrafiltration ultrafiltration  69  111.2.5.  Isolation  I I I .2.5.1.  Cell-Free  About least and  two  made  400  into  at  added, 5°.  Another  rotor.  RC-2  a  kept  time  2 mg  120  mL  of  rotor  at  most  monooxygenase  of  of  was  using  g  mode a t column T  (see  buffer  a  flow  (150  g,  Table T-100  of  5x15  cm)  II 1.1). for  a  0.5h  A  (ca.  12  for  minutes  an  The  180  mL/h  again  and  from out  anion  cell-free applied a  column the  at the  in  a  GSA  enzyme  centrifuged  to  obtain extract  DE-52  the at  a was  was  other  25°,  unless  exchange  column  extract i n the  then  effluent  derived  descending  anion  previously equilibrated This  h)  P-450  carried  to  were  a  the  8  atmosphere.  P-450  was  for  and  with  cell-free  argon  This  minutes  from  was  two-step  bacteria  rate  RNase  15  debris  This  (5°)  added  liquid  cytochrome  from  of  1 and  at  temperature  room  for  30  for  T-buffer.  then  cell  Cytochrome  procedure.  400  rpm  under  chromatographic about  of  cold  rpm  solution.  components  specified,  room  overnight  were  the  cloudy  of  at  frozen  c e n t r i f u g e equipped  15,000  I l l .2.5.2.' S e p a r a t i o n  been  mL  DNase  10,000  a l l times  Separation  of  continued  at  coloured  320  i n the  T-buffer  resultant  ice at  otherwise  each  had  thawed  with  stirred  stirring  removed  pink on  then  which  were  paste  the  The  SS-34  clear  bacteria,  refrigerated  This  extract. on  of  centrifuged  Sorvall  P-450  Extract  creamy  was  and  Cytochrome  in dry-ice,  a  which  mixture  g  weeks  suspension hours  of  exchange  with  buffer  washed  with  discarded.  70  Subsequent the  effluent  ascending  (300  g,  with  mode  5 x 3 0 cm)  buffer  were  well  P-450  band  which  band.  At  this  600  was  pink  bands  the  pink  of  two  columns  The was  made  ammonium  1mM  on  gentle  g  of  over  ice at  which  NH„OH  (NHjJzSO,,  tubing time  cytochrome  putidaredoxin  from  T-100  yellow  Tand  DE-52 c o l u m n  was  bands  entered  the  column  was  second first  was  passed  required  to  discarded  through  in  for  a  6  ice prior running  m to  the  Fractionation  solution  from  grade)  period  was  was  bands  hours.  (enzyme  To  pink  immersed  D-(+)-camphor  a  of  eluted  band  enzyme  separate  pink  elution  putidaredoxin  brown  first  and  band  a l l times  stirring.  concentrated 30  with  The  elution  14  the  column  The  three  gradient  the  total  of  applied in  exchange  yellow  the  yellow  Sulphate  enzyme  sulphate  saturation) kept  about  the  further  band.  diameter The  to  P-450  the  front  order  Ammoniurn  crude  in  in  yellow  was  time  salt  the  then  b u f f e r T-50.  until  linear  and  5°.  with  a  brown  was  DE-52 a n i o n  by  cytochrome  at  column  followed  the  small  collection  this  running  when  The  At  was  column,  continued.  coil  was  started  disconnected  second  continued  point  from  a  this  equilibrated  separated. band  and  to  T-100  reductase  second  from  of  30  under  an  maintain slowly  added.  by  a  second  and  200  minutes. argon  to  the  mixture  DE-52  column  fractionated  adding  constant  added  The  the  The  with  g/L  (36%  mixture  was  atmosphere pH  value,  mixture was  with  1 mL for  stirred  of  each for  71  another  30  minutes;  minutes the  and c e n t r i f u g e d  resulting  precipitate  was  by  140 g / L o f  adding  same  white  discarded.  procedure.  The 2  and  precipitate  resuspended  p-100 was  was  containing  stored  purified  under  the  8 mM argon  yellow  at  by g e l f i l t r a t i o n  5°  was  was  supernatant  This in  f o r 10  a  discarded. of  enzyme  Schlenk  chromatography.  using  collected  volume  crude  P-450  precipitated  (60% s a t u r a t i o n )  i n a minimum  camphor.  rpm  apo-cytochrome  precipitate  centrifugation  10, 000  supernatant  (NH„) SO»  The  at  the by The  buffer solution  tube  until  72  III.2.6.  Gel-Filtration The  Purification crude  incubated  100  mM  samples  two  portions  run  at  200  5°  the  The  by  inspecting  the  pink  applied column  gravity  the  t-o t h e  second the  spectroscopically  m o l e c u l a r weight  mg  of  g  of  protein  the  of  45,000  ( c a . 3 mL  of  a  so  divided  less  were  two  avoid and than  selected  60-70% after  of  being  were  fractions  yield  solution)  The  into  to  about  samples  P-  enzyme  equilibrated  then  of  this  which  were  calculated  D a l t o n s amounted  1 mM  and  aliquots  fractions  usual  40-120  columns.  included;  enzyme  to  mM)  column  middle  prior  A l l  order  that  and  size  head-pressure  first  The  pure  was  DTT  DTT)  the  were  those  column.  most  to  in  was mL  mM  ( c a . 0.5  columns  a n a l y z e d . The  a  mL  density  2  (10  bacteria  elute  about  contained  from  colour  to  T-100  flow with  fractions  with  (particle  applying  Both  mM  respectively.  1-2  of  50  temperature  separately  columns.  enzyme  concentrated  g  purified  under  mm.  400  made  G-100  with  before  from  P-450  room  buffers,  concentrated to  and  overloading  at  equilibrated  solution  were  Sephadex  ultra-filtration  enzyme  Cytochrome  samples  camphor)  were  of  minutes  by  columns  (8  by  enzyme  f o r 30  purification micron)  Chromatography  from  to about  135  isolated  from  400  another  two-step  bacteria. The  Removal  camphor  gel-filtration Sephadex  G-10  at  of  Camphor  substrate  was  Substrate removed  by  column  chromatographic  5°. Pure  cytochrome  P-450  procedure solutions  using were  73  made 30  50  minutes.  Sephadex buffer  with  adjusted effluent second  Camphor  G-10  was so  thiol  special  care  G-10  column  isolation  taken  samples  avoid  residual  bound  absence  of  an  optical  presence  of  50  mM  over  the the  camphor,  partly  prepared  the  24  this  camphor,  with  and  to  removal  ion  (see  of  enzyme  glassware as  this  manner as  was  period.  The  on  buffer  and  'tubing  sometimes  led  enzyme.  The  indicated  Section  the  camphor,  demonstrated  at  a  P-100  remove of  a  T-100  chromatographed  absorption shoulder  potassium  mL  hour  substrate-bound  in  on  with  1.0  for  head-pressure  a  exchange  in contact with of  about  then  During to  gel-filtration  equilibrated  buffer  reagent.  to  was  temperature  equilibrated  and  ran  room  by  Only  run  column  solution  was  cm)  DTT.  the  came  apparent  1OmM  a  removed  (1.Ox 20  that  which  enzyme  then  f o r each  effect  excess  incubated at  used  enzyme  to  and  was  column  Sephadex  order  the  DTT  containing  solution  in  mM  391  by nm  in  III.2.7.).  no the the  74  111.2.7 . S p e c t r a l A n a l y s i s The  purity  of  cytochrome  estimated  conveniently  coefficient  of  heme  the  protein  dodecyl  (SDS)  electrophoresis recordings of  Weber  from  and  spectral  by  the  280  3  91:A  700  cytochrome  nm  2  of  1.63.  than so  o  P-450  ( g r e a t e r than  ratio  of  Purified  1  95%  nm  Cary  in  a a  designed  to  handle  used.  cell  rubber Glass  cell  attachment volume  of  but  peak non  at  method  absorption  indicate 99%  391  that  homogeneous  nm  to  the  crystallized  have  an  special  (1x1x5 glass  to the  cell  enzyme  an  peak  samples  absorption  a  cell  recorded  B-14  A  (20 high-  sidearm  vacuum-gas tonometer  model  solutions  c o n s i s t e d of  tubing  the  were  tonometer  cm)attached  fitting.  with  spectra  spectrophotometer  tonometer  septum Co.)  protein,  gel  ratio,  1.45.  r  through  the  homogeneous  temperature,  mL)  the  optical  heme  absorption  quartz  the  sodium  densiometric  using  the  Electronic  The  with  in  the  SDS-tube  of  scanner  of  e l e c t r o p h o r e s i s ) i s c h a r a c t e r i z e d by  absorption  greater A  of  gel  gel  combined  3  mobility  analytical  be  extinction  maximum  integration  automatic  Osborn,  SDS-tube  optical at  an  the  The  1  can  the  absorption  electrophoretic  gels.  by  data  crystalline  the  samples  correlating  electronic  with  sulphate  by  P-450  was  to cm)  clear  room  III.4)  rectangular reservoir  containing O-ring  socket handling mL.  at  (Figure  sidearm  vacuum  60  17D  250-  anaerobically being  a  a  from  was  a  silicon  joint  (Kontes  utilized  line.  (10  The  for total  1  gure  III.4  The  cell-tonometer.  cm  quartz  cell  76  Previously transferred with  argon  to  degassed. the  and  sidearm  evacuated  (ca.0.2  mL)  and  transferred  then  tonometer. state  of  The the  reduction out  by  (ca.  was  septum  sodium  at  314  reduced  enzyme  at  room  of  was  enzyme  nm  the  bubbling  due  to  was  the  temperature.  gas  mixed  cavity  to  the  of  a  ferrous sodium  P-100)  until  a  excess  by  through  the  flushed solution swirling  tilting of  the  the ferric  Stoichiometric  state  was  dithionite through  adding  the  was  was  the  carried solution rubber  s m a l l a b s o r p t i o n peak  recorded.  enzyme  by  dithionite. by  syringe;  then  reduced  enzyme  recorded.  removed  gas-tight was  then  buffer  syringe,  dithionite via a  gently  a  Stock  reservoir,  cell  mL)  repeatedly  a b s o r p t i o n spectrum  in degassed  dioxygen  complex  the  (4.8  and  times.  the  s m a l l amounts  using  appeared  to  to  buffer  reservoir  three  optical  the  adding  4 mg/mL  added  enzyme  of  P-100  Any  excess  s m a l l amounts  optical The  spectrum  carbon  conveniently solution  of  monoxide  formed  f o r 30  of  by  seconds  77  111.3  Determinat ion  Reaction  of  P-450  Pifferent  at  Carbon  111 . 3 . 1 G e n e r a l The  with  gas  pressure.  changes  as  a  described In  can  The  apparatus  was  butylphthalate  Hg  7.73X10~ 111 . 3.2  for = mm  2  for  Hill  as  and  carbon  P-450 by  a  measuring  of  at  observing  function the  plots  of  spectral  determining  log/log  a  the  has  been  5  for  the  (ca.0.8  addition mm  Hg),  with  manometer  pressures  data Hg,  reaction  changes  pressure  constructed  manometer.. P a r t i a l mm  gas  via  gas  (DBT)  the  conveniently  spectral  procedure  allow  CO  for  measured  absorption  to  of  to  be  previously." •  pressures  the  Cytochrome  s u b s t r a t e - f r e e cytochrome  constant  order  Substrate-free  constant  f u n c t i o n of  equilibrium  with  for  Informat ion  temperature  electronic  E q u i l i b r i u m Constant  Temperatures  reduced  the  the  Monoxide  equilibrium  monoxide given  of  a  an  small  partial  vacuum-gas  handling  incorporated  i n s t e a d of  of  CO  analysis  derived  of  i n mm  using  the DBT  the  using  density  were  freeze-thaw  usual were  di-nmercury  converted  formula,  mm  DBT  data.  Procedure  All  buffer  solutions  argon-saturated  before  100  added  b u f f e r was  system  evacuated  cell  was  compartment  and  then of  the  use. to  About  the  optical  refilled  transferred Cary  17D  4.8  with to and  mL  of  cell  argon the the  degassed  and  camphor-free  tonometer three  and  times.  thermostated base-line  Pthe The  cell  spectrum  78  recorded model  from  FK  250-700  constant  connected  to  nm.  For  temperature the and  line.  b e g i n i n g of  Before  temperature  baths  (±0.2°)  and  at  least  30  to  the  all  solution Then  line  as  and  was  this  cell  thermostated  taken  of  in  I I I . 2 . 7 and  and a  full  attached  to  of  to  the  bath.  temperature  (±0.2°),  Two  allowed vapour  to to  remove  the  p r e s s u r e of  the  substrate-free  gentle  swirling. in the  At  enzyme  in  again and  then  the  to  the  The carried  the  a  enzyme  >5  out  as  recorded. CO-gas  described The  copper  port  the  of  used  was  atmosphere  and  minutes)  before This  line in coil  constant  t o measure the  tonometer  cell  immersion  spiral  were  solution.  oxidized  handling  by  C O - l i n e and  cell  of  the  stoichiometric  temperature  the  (  placed spectrum  spectrum  argon  equilibrate  again attached  bubbles  circulation  at  was  for  was  thermometers one  temperature  stock  with  form  containing  spectrophotometer.The  evacuated  of  temperature  protein.  desired  water  temperature  the  to  was  taken  the  constant  t o d e n a t u r a t i o n o f 'the  the  water  the  mL  recorded.  enzyme  then  thermostated  Dewar  lead  enzyme  ' was  0.2  not  at  the  handling  the  required  tonometer  compartment  the  the  one  of  CO-gas  experiment,  anerobically  reduction  tonometer  to  cell  about  can  cell  each  the  Haake  utilized,  compartment  at  equilibrate  tonometer  substrate-free  Section  other  . The  were  cell  brought to  added  care  the  were  minutes  solution  times,  the  allowed  argon  enzyme  the  baths  special  spectrophotometer  t h e r m o s t a t i n g , two  other  the at  then  slowly  the  system  reading  procedure  the was  79  repeated was  three  converted  accuracy, the  to  The  equilibrate partially spectrum  a  from to  added  the as  and  spectrum.was This  of  gas  enzyme  published  there of  CO  were gas  to  and  was  prior  was  five  recorded  under  to  cell  procedure  in  order  to  the  equilibrium  was  and  back  using  different i n each  followed  investigate constant.  tonometer  at the  slowly to of  Once  the  the  CO-  the  same  CO-pressures  case.  of  (3  spectrum  to  in  carefully  allowed  recorded.  1 atmosphere  for  6  leaks  line,  the  readjusted  least  data  equilibrate  the  taken  obtained  no  was  handling  being  cell  spectra  recorded  general  temperatures dependence  the  recorded  pressure  spectrophotometer  the  At  that  3 minutes  pressure  above.  with  allowed  the  the  recorded,  vapour  amount  and was  for another  and  sure  small  pressure  of  checked  make  system  taken  was  value  and  carbonylated  procedure were  to  removed  and  The  Hg  Then  the  closed,  swirled  mm  in order  minutes).  line  to  apparatus.  admitted  was  times.  The  final  CO. four  different temperature  80  111.4  Determination  Reaction  of  of  Dioxygen  E q u i 1 i b r ium  with  Constant  Substrate-bound  for  the  cytochrome  P450cam 111.4.1  General  The with  Informat ion  equilibrium  substrate-bound  experimental  previous  of  CO  as  buffer  the  111 .4 .2  volume  ( 30  spectrum  solution 6  M  using  used  to  of  was  that  50%  dioxygen  bath  an  for  the  in  the  system  at  discussed the  (Figure  constant  by  used  equilibrate  compartment  of  estimated  constant,  slush  cell  was  and  was  mL  was  of  then  the was then  the  III.5)  temperature  was  not  was  baths  necessary.  diethyleneglycol  bath  buffer  as The  in P-buffer.  i n the  to a  Cary  final  out  to  slush  as  the from  7  and  to  given  the  the  list  N  about  tetrachloride/liquid  o-Xylene/liquid  N  2  N  2  2  stoichiometric III.3.2.  temperature  (±  below.  -13°  2  as  enzyme  Temperature  Benzonitrile/liquid  of  base-  spctrophotometer  in Section  desired  2/3  the  c o n c e n t r a t i o n of  Slush  Carbon  filled  substrate-bound  r e c o r d e d . Then,  carried  was  solution  III.3.2. Stock  added  cooled  slush  the  recorded  spectrum  suitable  the  ) with  _ in Section  reduction cell  to  use  cell-cavity  described  1CT  similar  reaction  Procedure  The  line  a  the  special  solution  be  order  temperatures  used.Therefore, well  In  f o r the can  binding  section.  sub-zero  P-450  procedure  determination  its  constant  -23° -29°  The 1°)  81  Serum cap  To vacuum l i n e  Dewar f l a s k  Constant temperature  Optical  cell  F i g u r e i n . 5 1 0 cm p a t h - l e n g t h o p t i c a l  bath  Quartz windows  cell  (Slush Bath).  82  The  temperature  addition  of  partial  pressures  pressure  gas handling  and made  small  amounts  the corresponding to record  species  under  was  of  0  maintained  of l i q u i d were  2  line  2  then  as d e s c r i b e d  spectra  the spectrum  1 atmosphere  N  were  constant periodically. added  of the f u l l y  a  low  III.3.2,  Attempts  formed  the Small  using  in Section  recorded.  of dioxygen  by  were  oxygenated  (see Section  IV.4).  83  REFERENCES  1.  I.C.Gunsalus,G.C.Wagner,Methods Enzymol.,52,166(1978).  2.  E.S.Lenox, V i r o l o g y ,  3.  K.Weber,  4.  D.Dolphin, Res.  5.  6.  J. Biol.  B.R.James,  Commun.,  88,  Chem.,  244, 4 4 0 6 ( 1 9 6 9 ) .  H.C.Welborn,  Biochem.  Biophys.  415(1979).  D.V.Stynes,B.R.James,J.Am.Chem.Soc.,96,2733(1974).  Handbook Rubber  7.  M.Osborn,  J_, 1 9 0 ( 1 9 5 5 ) .  of  Chemistry  Publishing  R.E.Randeau,  Co.,  and P h y s i c s ,  41st Edn, Chemical  Cleveland(1959-60)p.2326.  J . Chem. E n g . D a t a ,  11 , 124( 1 9 6 6 ) .  CHAPTER  RESULTS  AND  IV  DISCUSSION  85  IV.1  Growth.of The  Pseudomonas  monooxygenase  bacterium  Pseudomonas  biological  model  studies  present  used  to  several  to  the  provided  cytochrome  release  to  the  from  a  definitive physical  P-450  mediated  systems.  strain  strain  In  1  786  was  786  possesses  PpG  1 which  was  by  enrichment  on  are  the  generation  shorter  bacteriophage,  hydroxylase providing  the  and  strain  parent  soil  by  in  h y d r o x y l a t i n g monooxygenase  advantages  lysis  the  thereby  camphor  putida  P-450cam. The  these  resistance  autolysis,  present  biochemical  Pseudomonas  over  isolated Among  1  has  the  the  advantages  camphor. time,  study,  cytochrome  originally  786  system  putida  to  Strain  r e a c t i o n s o c c u r r i n g i n mammalian  isolate  component,  enzyme  extensive  relevant  hydroxylation the  for  putida  proteins  easy  and  the  by  access  D-(+)-  ability  freeze-thaw  to  the  P-450  the  ability  enzyme. In  order  metabolize it  was  plates  for  camphor,  necessary containing  the and  hence  to  maintain  camphor  Failure  to  do  ability  to  multiply  subsequent It media for  was  depend  the  flask  long  so  growth. stages,  in  In  as  the  where  case  strain  the  only  the  glucose  carbon  and  trends energy  L-broth  source.  media,  of  500  glutamic  acid  the with  2  the  source  and  agar  losing  curves.  to  enzyme,  in minimal  saturated  pH  and  P-450  bacterium  growth  the  of  the  the  i n the  carbon  retain  produce  in  that  basic  to  camphor  lag-phases  the  to  results  observed on  bacteria  culture provided  mL  shake  were  used,  86  respectively, growth  on  decrease the  during  has  metabolism  is  antifoaming  air  The  build-up which the  exit  in  but  the  during  the  was  order  bacterial  this  also the  the  pH  to  clear during  growth  14 to  of  the  use L  fermenter  clog  cut  of  and  the  by  the  off  This  process,  growth,  resulted  use  of  an  via  rubber  in  external tubing  to  collect  the  foam  cells)  served  as  an  resulted cells  no  the  thus  fermenters in  due  while  the  filters  the  pink  of  of  overnight  removed),  fraction  in  fermenters.  cells;  pH  substrates.  part air  the  The  be  in  despite  c u l t u r e medium  bacterial  contained  considerable  due  in to  the  lack  death  of  of  a  aeration  agitation. The  at  the  of  to  increase  out',  caused  to  pH.  encountered  later  filters  alternativ-e,  the  the  connected  decrease  i s known  organic  'foaming  pressure  while  3  for  other  happened  of  air  (which  given  the  a  increased,  metabolites, "  at  high  death  in  metabolism  difficulty  to  of  media  acidic  the  agents,  usually  (with  of  foam  container,  of  the  supply  the  resulted  been  basic  bacteria  stage.  of  camphor  accumulation  The  and  pH  camphor  explanation the  the  least  duration twice  procedure, closely. amount  of  reported.  a  the  growth  as  that  a l l  consequence  enzyme 1  long  although  1  As  as  of  isolated  the  of was  the  procedure given  in  was the  found  generally  growth smaller  be  literature  i n s t r u c t i o n s were lengthy  to  followed  period, than  the that  87  IV.2  P r o p e r t i e s of Cytochrome  IV.2.1  Substrate-Bound  The  absorption  substrate-bound  are given  absorption  maxima  millimolar together The (protein nm  Cytochrome spectra  cytochrome  II.2.6.1)  P~450cam  of the various  P-450cam  in Figure  ( max),  well  data  (visible  391  nm  (Soret  peaks).  The  with  the published  absorption  at  280  nm,  the  chain.  slightly the  (±  On amounts enzyme  protein  of small  reducing of  the  sodium  solution  generation  the  through  t h e enzyme  yellow  colour  low e n e r g y  discrepancy due  to  from  the  in  reduced  (^max  light at  542, a n d  nm  with 314  and  formed  of  excess  the  groups i n varied  P-450.  the c o l o u r of pink  with  nm;  the  544  by  seconds,  Soret)  f o r t h e 365 nm  for  as expected.  of a  split  643 agree  peak  to light  408  few  nm  stoichiometric  nm),  brown  unchanged  for a  a t 280  p r e p a r a t i o n s , due t o  a t 365 a n d  interference  of the  presented  except  absorptions  values  and  values  o f t h e 280  enzyme,  solution  bands  mM  E  data,  state  peaks  remains  with  absorbs  and 510,  enzyme  oxidized  of absorption  of  enzyme  amounts of apo-cytochrome  dithionite  absorption  are  i s due t o a r o m a t i c  intensity  changes  complex  and  which  5%), i n different  presence  II.2.5  corresponding  mM)  e  calculated  well  The  the  (  peak),  reasonably  protein  as  pure,  i n Table IV.1.  oxidized, substrate-bound peak),  (Sections  coefficients  literature  s t a t e s of  I V . 1 . The w a v e l e n g t h s  as  A  extinction  with  P-450cam  and peak  The  CO-  bubbling  CO  has a  445 nm 550 (Table  dithionite  golden  (near nm.  uv The  IV.1) i s in  the  1.0  oxidized  0)  o c  reduced  (0  reduced-CO  M O  5  Dithionite absorption  oo  300  Figure  IV.1  400  Various s t a t e s of pure,  —I 500  substrate-bound  6 0 0  Wavelength  c y t o c h r o m e P-450cam.  (nm)  7 0 0  oo oo  89  Table  IV.1  Absorbance  State  Data  (a)  X  Oxidized  for Substrate-Bound  Expt. Prep.  e  max 280  Reduced  Reduced-CO  23+2°;  e  species  mM at  (b)  From  ref. 5  (c)  From  ref. 1  X  mM  max  Prep.  e  mM  -  -  280  63.3  391  101  391  102  510  13.0  540  11.2  12.2  510  542  10.9  540  -  643  4.2  645  -  645  5.4  408  80.7  408  83  408  86.5  544  14.5  542  15.2  542  16.0  365  51.1  -  -  364  60.8  446  1 19  446  120  550  14  1 20  in  13.8  P-100  calculated 445  Prep. (c) L i t .  e  max  P-450cam.  510  550  Determined  X  mM  1 02  445  (a)  (b) L i t .  81.2  391  Cytochrome  nm.  550  13.0  buffer,  assuming  e  pH  7.4,  mM=120  8 mM for  camphor  at  reduced-CO  90  literature in  preparation;  the present  work  and t h e s m a l l e r  i s considered  more  e  value  accurate.  obtained  91  IV.2.2  Substrate-Free  The  absorption  substrate-free shown  in  Table  (Tables  IV.1  and  states.  However,  remarkably  states  (Section  max  values  e  with  of  pure,  II.2.6.2)  a n d mM  are given  are  f o r the  the l i t e r a t u r e  the  different  rapidly  similiarities states)  of  present  spectral  make  substrate-free Purity  The  data  1  of  no  the forms  is  used  have  IV.3).  The  strong  Because (and  trace  very  reduced-CO  and  of  the  reduced-CO  substrate-bound  that  enzyme  have  (Figure  states  or glassware  and S t a b i l i t y and  having  absorption  preparations  and  enzyme  of reduced  was  an  ratio 1.00  5  ,  o f t h e Enzyme  purified  substrate-bound  homogeneous,  forms  II.4.2.1).  and  various  enzymes, i t camphor  was  i n the studies  of  enzyme.  isolated  states  properties  sure  i-n t h e b u f f e r s  both  reduced  to substrate-free  substrate-free  of  substrate-free  that  i n the  (Section  to  and  oxidized  in spectra  essential  characteristics  IV.2) r e v e a l s spectra  o f camphor  occurs  The  x  substrate-bound  absorption  IV.2.3  the  of the s p e c t r a l  similar  was  of t h e v a r i o u s  IV.2.  of the  binding  P~450cam  P-450cam  IV.2;  preparation  Examination states  spectra  cytochrome  Figure  experimental in  Cytochrome  (Sections  enzyme  samples  absorption for 1.13  2  ,  Preperations  ratio  some 1.37  6  were A  3  of ,  9  1  and  at  80%  :A  the  1.45  II.2.5  2  least B  0  =  1.14.  literature  (crystalline  sample) . 1  All  substrate-free  enzyme  samples  were  of the  same  Dithionite  F i g u r e IV.2  absorption  Various s t a t e s of pure, s u b s t r a t e - f r e e cytochrome P-450cam.  93  Table  IV.2  Absorbance  Data  State  for Substrate-Free  (a)  X  Expt  e  max  Oxidized  Reduced  Reduced-CO  species  emM  at  e  max  (c) L i t .  X  mM  max  Prep.  e  mM  -  -  280  68.3  360  41.0  -  -  360  36.7  416  117.9  418  1 04  417  535  9.7  535  10.3  535  11.6  568  9.3  570  10.4  569  11.9  408  78.9  408  69.0  408  76.7  540  12.5  540  13.5  540  15.1  447  104  447  1 15  365  Determined 23±2°;  X  Prep.  107  550  at  mM  (b) L i t  P-450cam.  278  445  (a)  Prep.  Cytochrome  120 12.5  in buffer  P-100,  c a l c u l a t e d assuming  445  (b)  From  ref. 5  (c)  From  ref. 1  nm.  12.  550  pH  7.4,  emM=120  no  550  camphor for  120 14.3  added,  reduced-CO  95  order  of p u r i t y  they  were  than  48  P-420  as the substrate-bound  prepared.  hours  caused  species (Figure  samples  for  prepared  on a d a i l y  the  could  formation  enzyme  could  temperature  IV.4).  the  be  P-450)  of  basis. stored any  stored  without  The a t 5°  P-420  from  argon  which  f o r more  of d e t e c t a b l e amounts of as  seen  Therefore,  equilibrium  be  a t 5° u n d e r  the formation  (denatured  spectrum  purified,  But, storage  enzyme,  in the absorption  substrate-free  ligand  enzyme  binding  studies  were  substrate-bound  enzyme,  once  f o r over species.  indefinitely  detectable loss  of  6 The  at  months  without  substrate-bound liquid  purity.  nitrogen  Figure  IV.4  P-420 s p e c i e s  formed i n s u b s t r a t e - f r e e c y t o c h r o m e P-450cam  soluti  97  IV.3  Determination  of  the  Temperatures  for  Substrate-Free  Cytochrome  IV.3.1  Acquisition  Equilibrium substrate-free experimental At  a  fixed  pressure  was  decrease 445 at  were  nm  of  peak  373,  the  experiments  The  as  from  IV.3)  peak  of  in  using  yeilds  P w  A  Q  from  the  value  changes in Figure  (7-8  10 "  mm  Hg,  Raw  Appendix  IV.5.  M),  6  the  resulting and  an  for  observed  given  (reduced)  the  formation  2  the and  ratio  familiar  hence  present as  of at  (A-A  0  of  CO  in  a  increase  in  were  observed  data  from  a l l  the  ( i ) ,  along  with  the  of  the  the a  the  P-450cam  K  given  CO where, at  absorbance  of  Fe(Il)-CO  =  absorbance  of  F e d I)  II.2)  log/log  plot  enzyme  a  to  the  pressure  )/(A -A),  =  (Reaction  can  be  which  value.  Fe(II)-CO  a o  CO-complex  Hill  CO  J  A  with  different  mean  spectral  nm.  cytochrome  determined  expressed  a  CO  III.3.  three  Isosbestic points  583  given  by  data.  substrate-free  complex  give  are  of  estimated  least  to  0.4-760  nm  and  are  degree  The  at  The  binding  in Section  concentration  408  466  the  given  out  (CO-complexed).  430,  processed  were  (Table  varied  Carbon  Pifferent  Pata  P-450  constants.  enzyme  of for  carried  no.5  of  values  temperature,  equilibrium  At  Monoxide  obtained  each  experiment  Binding  Treatment  constant  data  for  and  at  P-450cam  cytochrome  determinations the  the  E q u i l i b r i u m Constant  known when  P  P c o  C Q  =0  decarbonylated ( P  c  o  ^  c  a  n  D  e  Figure  IV.5  Spectral  changes  observed  f o r the  P.  /  0  experiment  no.  5.  99  A ^  absorbance  Clearly,  A-A  0  of  fully  a. [ F e ( I I ) - C O ]  Since,  where,  are K  Log  (A-A )/(A  K'=  x.K  C Q  =Log  o o  -A)=Log  calculated  the plot  of  be  using  the the  i n water  r o  from  mm  III.3.1.  The  DBT  with  U.B.C.  the  0.90-1.15; data  points  .  a  vs. log  theoretical  the  The  K  in  temperature.  equilibrium;  =1/X.P  C  O  and  P  gradient  when  corresponding  (M  - 1  C O  ) values  PQ-,  can  be  values Hg  plotted  The  a t 445 as as  gradients  resulted  calculated nm.  in Figure  from  the  values  were  in  Section  IV.6 u s i n g  from  the  at  i n a much  were i n  , removal  to  contain  least more  the  computer  of a l l the p l o t s  instances  that  P^-,  described  (considered  the c o n d i t i o n  remained,  were  available  in certain point  1 / 2  values  "ace:graph"  at  applying  2  t o mm  were  facility  experimental  c o  -A)  converted  data  expressed  equation,  0  program  K'  0  line  P , y  absorbance  data  Log  co  [CO]  l o g (A-A )/(A^-A)  f o r the simple  corresponding  error),  P+  o f CO  straight  l o g (A-A )/(A  range  - Log  at the reaction  K  computer  -A)  0  would  The  O D  o r , l o g ( A - A ) / ( A ^ - A ) =0 ,  _  value  Hg)  a  to unity  =Aoo A  0  the s o l u b i l i t y  give  following  and  M/(mm  should  (A-A )/(A  0  Therefore,  the  derived.  Log  x=  0  , and  =[Fe(II)-CO]/[Fe(II)][CO],  relationships  A-A  Fe(II)-CO  oc[Fe(II )] .  A^-A  equal  formed  four  of a  an  large other  satisfactory  100  101 correlation  of  regression  data,  analysis.  experiments  are  calculated  from  value  given  converted  to  published  data  biological  for  7  with  sensitive  van't  the  constant  (K  solubility  of  c o  value  1 / 2  and  the  mean  values  ,M  _ 1  )  CO  were  values in  using  water  at  study  ligands  is  the  determination  of  and  AS .  case  the  AH  0  van't  Hoff  plot, the  thermodynamic  for  in  measurements  C  Q  computer  In  of  also  1/T  ;  were  of the of  parameters  although  8  are  program  parameters  the  such  plots,  v s .  reactions  highly  available.  9  Figure  IV.7)  mentioned  above,  calculated.  The  relationship, K  a  and  (log K  of  0  determination  via  using  Log  free  P,^  P  P,^  the  Hoff  thermodynamic  The  The  microcalorimetric techniques  and  higher  recorded  a l l  of  been  obtained  (usually  w  linear  aspect  systems,  was  observed  the  for  ( i i ) . Each  IV.3).  parameters  usually  gradient  plots  least-squares obtained  Appendix  equilibrium  thermodynamic  allows  plots  (Table  important  hemoproteins  The  in  to  temperatures.  An  has  The  these  calculated  various  according  =  -(AH°/2.303R).1/T  plot  AS  of  from  0  these are  0°-40°)  log K  the  1/T  AS°/2.303R to  y-intercept.  plots done  vs.  +  owing  in  because  a  of  to  Good the  narrow  the  give &H°  from  the  linearity  is  fact  that  temperature  instability  ,  of  the  range  proteins  8  at  temperatures. AH  0  value  cytochrome  for  the  P-450cam  CO  was  binding estimated  to to  reduced be  -18±1  substratekcal/mol  102  Table  IV.3  The  Equilibrium  Substrate-Free  T(°C)  4.0  12.0  18.0  24.0  Pimm 1/2  #  Constant  Cytochrome  H g ) Mean  P^  Values  K  C Q  (M* )  0.372  2  0.423  3  0.398  1 .45x1 0  4  1 .38  5.07x10  5  5  1 .32  5.30X10  5  6  1 .57  4.63x10  s  7  2.63  2.95x10  s  8  2.69  2.88x10  s  9  3.02  2.57x10  s  10  4.68  1.57x10  s  1 1  4.47  1.65x10  s  1 2  4.62  1.59x10  s  1 .55X10  1 .40  2.78  4.59  CO  Binding  to  P-450cam.  1  0.398  for  1.37X10  Mean  Log  K  C O  l/TxlCT  6  6  1.46x10  s  6.1644  3.608  5.00x10  s  5.6990  3.507  2.80x10  s  5.4472  3.434  1.60x10  s  5.2041  3.365  6  3  103  1 0  3.36  3.4  3.44  3.4B  3.52  3.56  3.6  3.64 1/T*10  Figure  IV.7  The  van't  Hoff  plot.  104  The  AS  M). the  0  value  Table CO  horse  IV.4  Mb,  sheep Given  -37±4  cal/mol.deg.  summarizes  binding  compounds. study  was  to  (standard  published  thermodynamic  substrate-bound Hb  also  ( p e r mole are  the  of  cytochrome  heme),  values  f o r s u b s t r a t e - f r e e cytochrome  state  and  P450cam.  data  1 for  P-450cam,  t o some  determined  of  in  model this  105  T a b l e IV.4  K  c o  ,  and  AH  and AS  0  Models  0  Values  f o r CO B i n d i n g t o Some  of Cytochrome  System  K  c o  P-450cam  P-450.  AH  ,25°  (M-  1  Hemoproteins  )  0  (kcal/mol)  1.3x10  5  2.5x10  7  AS  Ref .  0  (cal/mol.deg.)  -12  -17  -12.6  -7.4  1 1  (sub.-bound) Horse  Mb  Sheep  Hb  -  -16  -  8  Human  Hb  -  -17.7  -  9  Model*  (a)  1.3x10'  -16  -35  1 0  (b)  2.8x10  3  -19  -48  1 0  1.3x10  5  -18±1  -37±4  P-450cam (sub.-free)  *  8  this work  Fe(II)Pp(IX)DME-BuS  (a)  in a polar  medium  (b)  i n a non-polar  complex (N,N-dimethylacetamide)  medium  (toluene)  106  IV.4 of  Comparison  Other  of  Hemoproteins  Structural reveal  that  the  CO  unit  to  distal  the  systems,  normal  to porphyrin  that  in  the  to  kcal/mol  to  from  value  IV.4).  reverse  considered  to  conformational As  termed been  CO  been  study  of  e n t h a l p y and  i n the  relatively  rigorously  est  minor. lished,  AH  1 7  lowered  the  which  binding  i s at  least  compared  0  terms  to  to  the  variety  of  the  system  feeling  this  (Table AS  0  exist  e n t r o p y change protein  phenomena'. effect'  so  that  under  in  a and  reactions It  has  produces the  net  investigation  principle  i s that  is  1 8  there  1 8  'compensation changes  of  5  the  differences  chain.  between  Although the  and *"  1  effect'  P-450cam  Rajender,  entropy  f r e e - e n e r g y of  are  that  in  protein  and  the  proposed  s u b s t r a t e - b o u n d system  i n the  that  interactions  i s linear  steric  show  mainly  a  the  protein-free  affinities  observed  Lumry  from  bent.  the  within  demonstrated  change  unit  has  i n terms  relationship  change  Fe-C-0  'enthalpy-entropy compensation  parallel  is  by  the  cytochrome  due  changes  linear  enthalpy  be  trend be  proposed  specific  for  1 1  It  present  favourable  published The  to  those  hemoproteins  to  in  'distal-side  substrate-free  more  1 3  to  tilted  p l a n e , due  the  the  this  the  and/or  However,  1 2  Values  0  carbonylated  bent  expected,  Fe-C-0 u n i t  Results CO  due  of  plane.  AS  Systems  porphyrin  hemoproteins,  significantly  of  as  and  Model  is  residues.  model  causes  and  AH°  analyses  the  perpendicular with  Measured  is  not  expansions  and  107  contractions volume' of  of  the  of  water,  system.  concentrations transition produce same  which For  example,  the  AH  0  and  0  effects and  AS  value  0  in changes to  the  effect  reversible  ribonuclease A  parallel  hemoglobin  result  contributes  the  A(AH )/A(AS )  SCN .  protein  on  of  compensation  -  the  each  have  entropy  change  varying  ethanol  thermal  unfolding  that  i s to  give  rise  to  temperature.  Also  similar  been  with  'free  various temperatures  changes  0  at  myoglobin  at  of  i n the  noted  ligands  in  such  the  reactions  of  F~,  N3,  CN~,  apply  to  the  as  1 8  The  compensation  results  of  the  contribution  observed,  in  AG  of  the  the  study;  in  P-450cam  change  0  present  observed  substrate-free  the  hypothesis appears  the  the  large  case  compensates  overall  reaction,  to  negative  of  CO  for the  process  binding large  resulting  compared  to  the  entropy to  enthalpy  i n no  change  substrate-bound  system. Qualitatively, the  binding  the  substrate-free  presence  of  of  the  the  CO  more  fashion.  the  inhibition site the  of heme  causing  the  In  be  molecule the  CO  such  by  where  s u b s t r a t e must  iron.  may  fact,  observations  experiments  less-favourable  substrate-bound  substrate  perhaps  supports  to  system,  active-site, angular  observed  they  enzyme, due  sitting  conclude  l i e i n the  et  to  immediate  to the  to  bind  in  the a  indirectly  a l .  that  to  close  hypothesis  Peterson  for  0  compared  mainly  molecule a  AH  1  9  the  in  their  bonding  vicinity  of  108  The (Table the  IV.4)  fifth  ligand Hb  relatively may  Mb,  determined  for  term  comparison. system occur  and  the  (Table binding  of  the  to  number  of  at  sheep  that  protein  chain  P-450  to  bound  system  None  of  using  these  the  difficulties near  autoxidized  (according  Fe(III)  enzyme)  0°,  during  the  in  the  to  the  terms  of  Mb  changes to  the  much  higher  model  system  AH  as  0  P-450cam.  Therefore,  is linear  (or  the i t is  almost  so)  complex. data  the to  0°  determine  to  -30°,  given  were  in  2  not complex  reaction of  for  work, the the  the  II.3,  on a  P^  2  values,  substrateIII.4.  because  resolved. was  0  large  Section  successful  were  time  available  present  enzyme-0 to  the  in a  that  temperatures  in  results  procedure  experiments  for  this  made  from  available  compared  P-450-CO  ranging  the  binding  Fe-C-0 u n i t  were  However,  change  CO  PAH°  conformational  of  sheep  the  CO  thermodynamic  attempts  to  to  i s not  of  thiolate  substrate-bound  entropy  favourable  In  property  binding  P-450cam.  binding  system.  myoglobin  the  similar  minor  upon  to CO  for  system  small only  the  is  substrate-free  no  P-450  Hb  system;  The  the  are  temperatures  several  it  relatively  substrate-free  binding  indeed  the  that  There  for  that  reflect  CO  term  0  to  6"-donor  compared  than  is equally  conceivable in  AH  constant  weaker  for  constant. IV.4)  the  substrate-free  substrate-bound binding  from  binding  the  The  may in  The  favourable  or  CO  imidazole,  P-450.  i s more  entropy  result  ligand,  in  450cam  larger  formed,  to  experiment.  of At but  give  the  The  only  109  reported  P,/  value  2  determined  using  the  method  used  binding  constants.  gives  the  However, the  for 0  in  half  the  published  rather  present  were  IV.5;  At  lower  reaction used  Raw  was  shaken  to  higher Data  surface  over  bubble  rising  spectrum  the long to  altered  than  the  not 0  substrate-bound  AS  values)  cytochrome  the  time  the  the  report  as  same 75  CO  minutes.  autoxidation (Table  the  1 1  of  IV.5) in  the  values  more the  floated  1 h),  during  the  and  that  the  of  of  the  single  After  determination  P-450cam.  to  a  such  that  binding  was  recording  observed.  the  thus  slush-bath  (ca.  for  system  viscous,  bubbles  decided  autoxidation  co-solvent  considerably  for  was  determine  reported  -30°)  when These  was  0°  tonometer  determined  the  not  suitable 0  tO  surface  i t  of  at  cell  complex  became  of  0°  tes  of  but  gas.  were  enzyme  (iii)].  (-10°  absorption  attempts  AH ,  a  at  rates  form  period  unsuccessful  (and  to  added  points  was  r  most  prevented,  isosbestic  approach  that  i n Appendix  bubbles  a  noted  the  temperatures  mix  to  11.20,21,22  (ethyleneglycol/water) small  value  be  study  P-450cam-dioxygen  indeed  causing  P-450  autoxidation  inconsistent;  work  [Table  should of  to  conventional  present  It  substrate-bound  are  same  the  life  binding  2  of  a  the many  current the  dioxygen  P  1 / 2  to  110  Table  IV.5  Half-lives  of Autoxidation  Substrate-Bound  Cytochrome  pH  T  Reaction  of Dioxygen  Complex o f  P-450cam.  (°C)  Half-life  Ref .  (min.)  7.0  0.0  75  1 1  7.4  4.0  45  20  7.4  4.0  1 15  21  7.4  2.0  21  22  7.4  4.0  18  22  9.2  22  7.4  10.0  7.4  0.0  7.4  10.0  Unfortunately, available excess  on  of  covalently  P-450  a  porphyrins,  equilibrium model  prepared attaching  thiol  can serve  be  good  candidates with  considerations.  by  group  as a b u i l t - i n  P-450  for  studies  systems  work  6.7  this  work  kinetic  that  ligand.  recently a thiol  this  or  systems  'non-innocent'  the  comparison  no  20  in  data  do n o t u s e a 2 3  Collman  et  ligand,  o n CO v s . 0 the  vast  Mercaptan-tai1 a l .  to the porphyrin axial  are  2  context  2  3  so  appear  by that to  binding for of  steric  Ill  REFERENCES 1.  I.C.Gunsalus, (1978).  G.C.Wagner,  2.  S . A l b o n , M.Sc T h e s i s , (1983) p.198.  3.  H.E.Conrad, R.Dubus, I . C . G u n s a l u s , R e s . Commun., 6, 2 9 3 , ( 1 9 6 1 ) .  Biochem.  4.  J.Hedegard, (1965).  Chem.,  5.  J . A . P e t e r s o n , A r c h . 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Temperature: 4  ± Q±2°  Buffer: P-100  C  Vapour Pressure of Water: 79 mm DBT  P : 7.4 H  Experiment No. 1 Raw Data  Species  P  p  CO  total  Processed Data  P r  co  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A 0  A.-A  A-A 0  A— -A  Log  A _ A  o  A.-A  L  °8  P  ™ CO  1  -  76  -  0.177  2  5  80  4  0.327  0.309  0.150  0.180  0.833  -0.07918  -0.5098  3  10  89  13  0.416  1.01  0.239  0.091  2.626  0.4194  -0.002120  4  20  97  21  0.443  1.62  0.266  0.064  4.156  0.6187  0.2104  5  40  111  35  0.467  2.71  0.290  0.040  7.250  0.8603  0.4323  6  100  183  107  0.492  8.27  0.315  0.015  21.00  1.322  0.9176  Appendix ( i ) Equilibrium Data f o r CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  Buffer: _  4f0.2°C  P  Vapour Pressure of Water:79  P  H :  1 0 0  7.4  Experiment No. 2 Raw Data  Species  P  p  CO  Processed Data  total  P co r  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A  0  A.-A  A-A  0  A<« -A  Log  A _ A  o  Log P CO  A..-A  1  -  76  -  0.205  2  5  80  4  0.385  0.309  0.189  0.245  0.735  -0,1339  -.5100  3  10  85  9  0.475  0,696  0.270  8.155  1.742  0.2410  -0.1600  4  15  91  15  0.490  1.16  0,285  -.140  2.036  0.3087  0.0643 (?)  5  25  98  22  0.548  1.70  0,343  0.082  4,183  0.6215  0.2306  -  -  0.063  6  Appendix ( i ) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4+0.2°C  Buffer: P - 1 0 0 79 mm DBT pH: 7 . 4  Vapour Pressure of Water: Experiment No. 3 Raw Data  Species  P  p  CO  Processed Data  total  P r  co  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  A-A  o  A-A QD  (mm Hg)  A-A  o  -  74  -  0.150  2  5  79  5  0.350  0.387  0.200  0.210  0.9524  3  20  95  21  0.485  1.62  0.335  0.0750  4.466  4  40  lit  0.520  3.48  0.370  0.0400  9.250  -  -  0.560  A - A  o  Log P CO  A - -A  1  5  Log  -0.0212  -0.4129  0.6499  0.2104  0.9661  0.5414  Appendix (1)  contM.  Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 12+0.2°C  Buffer: P-100  Vapour Pressure of Water: 135 mm DBT pH: 7.4 Experiment No.4 Raw Data  Species  P  p  CO  Processed Data  total  p R  CO  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A o  A.-A  A-A o A<» -A  1  -  135  -  0.195  2  5  140  5  0.285  0.387  0.090  0.355  0.254  3  10  164  29  0.480  2.24  0.285  0.160  4  30  215  80  0.565  6.18  0.370  5  50  405  270  0.610  20.9  0.415  6  oo  -  -  0.640  Log  A _ A  o  Log P CO  A.-A  -0.5960  -0.4129  1.78  0.2507  0.3506  0.075  4.93  0.6931  0.7913  0.030  13.8  1.141  1.320  Appendix ( i ) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  1  2  Q  +  2  o  Buffer:  c  Vapour Pressure of Water: 135 mmDET  p _  l  0  0  7.4  Experiment No. 5 Raw Data  Species  P  p  CO  Processed Data  total  P r  co  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm tt^)  A-A 0  A.-A  A-A 0  Ao -A  Log  A _ A  o  Log P CO  A.-A  1  -  134  -  0.222  2  5  138  4  0.260 ?  0.310  0.038  0.600  0.0633  -1.198  -0.5100?  3  10  141  7  0.397  0.541  0.175  0.463  0.378  -0.4225  -0.2668  4  20  163  29  0.610  2.24  0.388  0.250  1.15  0.1909  0.3506  5  50  196  62  0.727  4.79  0.505  0.133  3.79  0.5794  0.6806  6  150  290  156  0.787  12.1  0.565  0.073  7.74  0.8887  1.081  7  OO  -  -  0.860  00  Appendix ( i ) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  Buffer:  Vapour Pressure of Water:  pH:  Experiment No. 6 Raw Data  Species  P  p  CO  Processed Data  total  p co r  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  p c o  (mm Hg)  A-A o  A-A GD  A-A o A— -A  Log  A  A  o  A^-A  Log P CO  1  _  135  -  0.190  2  10  153  18  0.372  1.39  0.182  0.210  0,867  -0.0621  0.1435  3  20  163  28  0.430  2.16  0.240  0.152  1.58  0.198^  0.3353  4  40  188  53  0.478  4.10  0.288  0.104  2.77  0.4424  0.6125  5  60  246  111  0.530  8.58  0.340  0.052  6.54  0.8155  0.9335  6  CO  -  -  0.582  Appendix ( i ) (cont'd) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 18± 0.2°C  Buffer: P-100  Vapour Pressure of Water: 200 mm DBT pH: 7.4 Experiment No. 7 Raw Data  Species  P CO  p  Processed Data  total  p  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  (mm Hg)  A-A o  A.-A  A-A o A<» -A  Log  A _ A  o  A.-A  Log P CO  1  0  192  -  0.220  2  10  200  8  0.325  0.618  0.105  0.445  0.236  -0.6270  -0.2087  3  20  219  27  0.470  2.09  0.250  0.300  0.833  -0.0792  0.3195  4  40  268  76  0.595  5.88  0.375  0.175  2.14  0.3310  0.7690  5  80  313  121  0.635  9.35  0.415  0.135  3.07  0.4877  0.9710  6  120  371  179  0.685  13.8  0.465  0.085  5.47  0.7380  1.141  7  oO  -  -  0.770  Appendix ( i ) Equilibrium Data f o r CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  Buffer:  Vapour Pressure of Water:  pH:  Experiment No. 8 Raw Data  Species  P  p  CO  Processed Data  total  p co r  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A o  A.-A  A-A o A - -A  Log  A _ A  o  Log P CO  A„-A  1  0  203  -  0.165  2  10  212  9  0.252  0.696  0.0870  0.333  0.261  -0.5829  -0.1576  3  20  313  110  0.482  8.50  0.317  0.103  3.08  0.4882  0.9295  4  40  403  200  0.528  15.5  0.363  0.057  6.37  0.8040  1.189  5  80  429  226  0.565 ?  17.5  0.400  0.020  20.0  1.301  1.242  6  oo  -  -  0.585  Appendix (1) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  Buffer:  Vapour Pressure of Water:  pH:  Experiment No. 9 Raw Data  Species  P CO  p  Processed Data  total  p co r  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  A-A 0  A.-A  A-A o A - -A  (mm Hg)  Log  A _ A  o  A.-A  Log P CO  1  0  205  -  0.225  2  10  216  11  0.310  0.850  0.085  0.295  0.288  -0.5404  -0.07040  3  20  221  16  0.348  1.24  0.123  0.257  0.479  -0.3200  0.09230  4  40  239  34  0.400  2.63  0.175  0.205  0.854  -0.06872  0.4197  5  CO  -  -  0.605  Appendix ( i ) (cont'd) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 24±0.2° C  Buffer: P_ioo  Vapour Pressure of Water: 290 mm DBT  pH: 7.4  Experiment No.IQ  Raw Data  Species  P  p  CO  Processed Data  total  p co r  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A o  A-A CD  A-A A«j  o -A  Log  A _ A  o  A^-A  Log P CO  1  0  250  -  0.290  2  15  266  16  0.326  1.23  0.036  0.500  0.0720  -1.1427  -0.1118  3  25  275  25  0.390  1.91  o.ieo  0.436  0.229  -0.6395  0.2810  4  40  286  36  0.490  2.82  0.200  0.336  0.595  -0.2253  0.4502  5  65  318  68  0.600  5.25  0.310  0.226  1.37  0.1373  0.7201  6  100  360  110  0.652  8.51  0.362  0.174  2.08  0.3182  0.9299  -  -  0.826  7  Appendix ( i ) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  Buffer:  Vapour Pressure of Water:  pH:  Experiment No. 12 Raw Data  Species  P CO  p  Processed Data  total  p 'co  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A o  A.-A  A-A o A A -A  Log  A - A  o  A»-A  Log P CO  1  0  263  -  0.190  2  10  355  102  0.415  7.88  0.225  0.125  1.80  0.2553  0.8968  3  30  454  191  0.455  14.81  0.265  0.085  3.12  0.4938  1.170  4  80  533  270  0.480  20.9  0.290  0.060  4.83  0.4842  1.320  -  0.540  5  Appendix ( i ) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature:  Buffer:  Vapour Pressure of Water:  P  H:  Experiment No. 11 Raw Data  Species  P  p  CO  Processed Data  total  p co c  445nm  A  wanted (mm DBT) (mm DBT) (mm DBT)  P  co  (mm Hg)  A-A o  A-A OD  A-A o Aaa -A  Log  A - A  o  Log P CO  1  0  272  -  0.220  2  5  277  5  0.260  0.387  0.040  0.510  0.0784  -1.1055  -0.4129  3  10  281  9  0.293  0.696  0.073  0.477  0.153  -0.8152  -0.1577  4  20  290  18  0.340  1.39  0.120  0.430  0.279  -0.5543  0.1435  5  50  356  84  0.543  6.49  0.323  0.227  1.42  0.1532  0.8125  6  80  387  115  0.590  8.89  0.370  0.180  2.06  0.3129  0.9489  -  -  0.770  7  Appendix  (ii)  Appendix -,—T  1  ( i i ) (cont'd)  1  r — i  1  -r~n— — — 1  r  "  •  -0 2  0?  OS  Experiment The  Hill  at  12°C.  •  10  u  log Pco  No.  log/log  •  4_  1  1  1  i'  r  1  L  2 2  -0.3  -01  i.<  r  6  —  p l o t s obtained f o r the CO-binding  ~~ to substrate-free  cytochrome  P-450cam  Appendix  ( i i ) (cont'd)  The H i l l l o g / l o g p l o t s obtained f o r the CO-binding to s u b s t r a t e - f r e e at 18°C.  cytochrome P-450cam  ro oo  Appendix (ii) (cont'd)  i««  Experiment No.  10  11  —  The H i l l log/log plots obtained for the CO-binding to substrate-free cytochrome P-450cam at 24°C.  Appendix ( i i i ) D e t e r m i n a t i o n o f r a t e o f a u t o x i d a t i o n o f t h e dioxygen compl of reduced, s u b s t r a t e - b o u n d cytochrome P-450cam. Raw Data Expt. No. : 1 Temperature : 0 ± 0.2 °C Buffer : P-100  A  Aoo-A  1.28 1.45  0.396  1.50 1.55 1.58 1.62  0.0560  A =1.68  - I n (Aoc-A)  Time (min.)  0.230  0.9263 1.4697  1.0 15  0.176  1.7323  20  0.130 0.100  2.0402 2.3025 2.8824  30 36  Expt. No. : 2 Temperature : 0 ± 0.2°C Buffer : P-100  55  ^=1.09  A^-A  -In(A^-A)  Time (Min;  0.790  0.306  1.1841  1.0  0.856  0.240  1.4271  7.0  0.920  0.176  1.7372  16  0.996  0.100  2.30 6  30  1.05  0.0460  3.0790  54  A  Expt. NO. : 3 Temperature : 10 + 0.2°C Buffer  A~=1.63  : P-100  Time (Min.)  Aoo-A  - I n (Aoo-A)  1.38  0.254  1.3704  1.0  1.48  0.146  1.9241  5.0  1.53  0.100  2.3026  9.0  1.57  0.0600  2.8134  14  1.61  0.0200  3.9120  24  A  


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