Electron Transfer in Biology and the Solid State - American Chemical

1 Overview of Biological Electron Transfer

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R. J. P. Williams Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford O X 1 3 Q R , United Kingdom

Electron transfer in biological systems uses metalloproteins to a very large degree. In this chapter, the properties of the metal ion sites are examined first. Subsequently the proteins are analyzed. The discussion is divided into metalloproteins for simple wirelike electronic conductor systems and proteins that couple electron transfer with other movements (e.g., of protons). Stress is placed on both structural and dynamic features of metalloproteins. It is shown that the metal ion and the protein are cooperatively adjusted in their properties, differently in different molecules, so as to optimize function.

A

HIS SURVEY O F L O N G - R A N G E O U T E R - S P H E R E E L E C T R O N TRANSFER i n b i

ology deals first w i t h s i m p l e electron-transfer proteins (Table I). T h e f o l l o w i n g d e s c r i p t i o n of the sites of the m e t a l ions i n these proteins is c o n s i d e r e d to be p r o v e n . 1. T h e ligands a r o u n d m e t a l ions i n s i m p l e electron-transfer p r o teins are selected to a i d electron-transfer rates; they r e d u c e the c e n t r a l charge o n the m e t a l or i n d u c e the l o w - s p i n rather than the h i g h - s p i n state of a m e t a l i o n . E x a m p l e s are sulfur, i m i d a z o l e , a n d p o r p h y r i n ligands for i r o n a n d c o p p e r atoms i n a l l the most i m p o r t a n t s i m p l e electron-transfer proteins. 2. T h e g e o m e t r y a r o u n d the m e t a l i o n i n s i m p l e electron-transfer proteins is generated b y the p r o t e i n fold. T h i s fold is so strong that there is m i n i m a l l i g a n d rearrangement o n change of charge. E x a m p l e s are the close-to-tetrahedral, entatic, state 0065-2393/90/0226-0003$06.25/0 © 1990 American Chemical Society

Johnson et al.; Electron Transfer in Biology and the Solid State Advances in Chemistry; American Chemical Society: Washington, DC, 1989.

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E L E C T R O N TRANSFER IN BIOLOGY A N D T H E SOLID STATE

Table I. Simple Electron-Transfer Proteins Electronic Circuit

Protein Cytochrome c Copper blue proteins Ferredoxins Cytochrome b 5

Mitochondrial periplasmic space Chloroplast chains A l l energy capture chains Reductases

of the b l u e c o p p e r proteins a n d the c o o r d i n a t i o n g e o m e t r y o f


l o w - s p i n i r o n i n c y t o c h r o m e c. 3. T h e m e t a l i o n sites are b u r i e d some 10 A i n t o the p r o t e i n so that adventitious reactions of the m e t a l ions w i t h s m a l l l i g a n d molecules cannot occur. T h e c o o r d i n a t i o n sphere is effectively c o m p l e t e a n d inviolate because the p r o t e i n f o l d does not relax readily. T h e m e t a l i o n sites are also r e m o v e d f r o m the solvent, water. 4. T h e conditions i n items 1, 2, a n d 3 generate i d e a l e l e c t r o n transfer sites. S u c h sites w o u l d p r o v i d e v e r y fast e l e c t r o n transfer i f the m e t a l c o o r d i n a t i o n spheres c o u l d c o m e i n t o contact w i t h each other (i.e., as i n s m a l l - m o l e c u l e o u t e r sphere reactions, i n w h i c h the u n i m o l e c u l a r rate constant for electron transfer w o u l d be close to 1 0 s i f there w e r e no t h e r m o d y n a m i c barriers). I n the context of the b u r i e d sites of the electron-transfer proteins, this means that a factor of u p to about 1 0 i n u n i m o l e c u l a r rate constant has b e e n forfeited i n o r d e r to gain positional d e p t h i n an insulator. T h e e l e c t r o n transfer sites are partially or c o m p l e t e l y h i d d e n centers. T h e rate constants n o w a c h i e v e d , a r o u n d 1 0 s" , are adequate for biological systems. 1 0

1

5

1

5

5. P l a c i n g the m e t a l i o n just b e l o w the surface of a p r o t e i n allows it to b e c o v e r e d b y a r e c o g n i t i o n zone so that the e l e c t r o n transfer distance is i n the range of 10 A b e t w e e n its c o o r d i nation sphere a n d one or perhaps two electron-transfer spheres of other p r o t e i n centers. 6. T h e m e t a l i o n is o r i e n t e d t o w a r d one quadrant edge of a r o u g h l y spherical p r o t e i n (e.g., the cytochromes a n d the b l u e c o p p e r proteins), so that electron-transfer distances to all o t h e r quadrant edges exceed 15 A . 7. T h e distance constraints i n i t e m 6 a n d the r e c o g n i t i o n surface constraints i n i t e m 5 ensure a h i g h l y selective route for e l e c trons i n a n d out o f the available s i m i l a r l y d e s i g n e d e l e c t r o n transfer proteins.


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Overview of Biological Electron Transfer

5

8. T h e r m o d y n a m i c control (the redox potential) is generated b y a complex set of factors, i n c l u d i n g the c o o r d i n a t i o n sphere atoms a n d geometry, the s u r r o u n d i n g p r o t e i n , a n d the ex posure to solvent. T h e s e potentials have e v o l v e d to c o n t r o l rates a n d to allow devices such as p o t e n t i a l droppers to connect electrons w i t h p r o t o n m o v e m e n t s . 9. A p a r t from these s i m p l e electron-transfer p r o t e i n s , w h i c h transfer electrons at l o w d r i v i n g force, there are two further groups of s i m p l e biological electron-transfer centers. I n v e r y fast l i g h t - i n d u c e d reactions there is often a considerable r e action d r i v i n g force, a n d of course the c e n t e r is h i g h l y e n e r Downloaded by 185.13.32.143 on July 5, 2016 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch001

gized. T h i s chapter w i l l not consider such

excited-state

electron-transfer reactions. A f t e r the i n i t i a l l i g h t absorption, electron transfer i n biological photoreaction centers b e c o m e s t h e r m a l transfer. T h e t h e r m o d y n a m i c d r i v i n g force is also important i n another group of p r o t e i n s , oxidases, i n w h i c h there is a large energy change b e t w e e n the redox

couples.

O n e t y p i c a l case is the reactions of oxocations (such as F e O ) i n s i m p l e oxidases (such as peroxidases) w i t h r e s t r i c t e d relax ation of the p r o t e i n . T h i s d e s c r i p t i o n applies to s i m p l e electron-transfer proteins that are i n v o l v e d o n l y i n a l l o w i n g electrons to go f r o m one site to another. T h e p r i m e examples are cytochrome c, b l u e c o p p e r p r o t e i n s , a n d m a n y i r o n - s u l f u r proteins. W e have called these proteins u n c o u p l e d electron-transfer p r o teins. A n o t h e r variety of proteins is i n v o l v e d i n electron-transfer reactions that are c o u p l e d to other events (e.g., o p e n i n g of clefts, c y t o c h r o m e P ; p r o t o n m o v e m e n t , c y t o c h r o m e oxidase). I n these proteins, w h i c h are often based o n h e m e centers a n d q u i t e different c o p p e r centers f r o m the b l u e c o p p e r proteins (see i t e m 6), there is e v i d e n c e for considerable change i n p r o t e i n structure associated w i t h change of oxidation state. 4 5 0

A t least two extreme sets of electron-transfer proteins have e v o l v e d . B o t h appear to be d e s i g n e d for the functions i n h a n d . W e can c o m b i n e X - r a y crystallographic data a n d h i g h - r e s o l u t i o n N M R spectroscopy to look closely at p r o t e i n structure a n d dynamics to evaluate the design. C r y s t a l lographic data reveal the o u t l i n e structure most accurately. T h e s e data can assist N M R spectroscopy to p r o v i d e a w i d e r perspective o n the d y n a m i c s . T o b e g i n , w e m u s t identify the function. F o r c o n v e n i e n c e , I separate three types of function of electron-transfer proteins ( F i g u r e 1). 1. S i m p l e electron transfer, as i n a w i r e made f r o m h o p centers (e.g., m a n y F e S centers i n electron-transfer chains ( F i g u r e 2). 2. Scavenger s i m p l e electron transfer, as w h e n a p r o t e i n brings



redox potential/mV NADH

_L_

-320 complex I (NADH-ubiquinone reductase)

FMN Fe-S

e l

Fe-S , c

site I

-305 -245

Fe-S 4


1 FAD

C

ATP

succinate

Fe-Sei

-20

Fe-S

Fe-S

•40

Fe-S

c s

\

complex II (succinateubiquinone reductase) + 30mV

p l

Fe-Sp,

ubiquinone « +30 sited

cyt.d*

ATP

cyt. b

T

Fe-S

+280

cyt. c,

•225

\ I

ATP

—

(+240)

R

cyt. c i site III

complex III (ubiquinone-cyt. c reductase)

+270 complex IV (cyt. c oxidase)

cyt. a

+200

cyt. a ,

+400

2Cu

T

•815

Figure 1. A representation of the electron-transfer chain of the mitochondrial inner membrane. There are three types of electron-transfer protein. Those described in the text as static simple electron-transfer proteins are represented by Fe-S and cytochrome (cyt) Ci. Simple scavenger electron-transfer proteins are represented by cytochrome c. The remaining cytochromes belong to coupled proteins. Some copper proteins belong to each of the three classes. (Subscripts identify different protein centers and are not of consequence in this chapter. Reproduced from ref. 28.)



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7

Figure 2. A typical fold of an Fe S electron-transfer protein, a ferredoxin, which shows afi-sheetstructure of parallel strands cross-linked by H bonds. (Reproduced with permission from reference 29. Copyright 1981 Japanese Biochemical Society.) 2

2

r e d u c i n g (oxidizing) equivalents f r o m m a n y centers a n d d e livers t h e m to one site b y diffusion o f the p r o t e i n i n a m a t r i x (e.g. c y t o c h r o m e c o f m i t o c h o n d r i a l electron-transfer chains). T h e s e proteins are similar to t h e first g r o u p , except for t h e i r surfaces. 3. C o u p l e d electron-transfer proteins that, after o r before t h e electron-transfer event, u n d e r g o considerable conformational adjustment so as to c o u p l e electron transfer w i t h p r o t o n m o v e ments, for example. F u r t h e r u n d e r s t a n d i n g o f these functions d e p e n d s o n a d e t a i l e d analysis o f p r o t e i n structure against t h e b a c k g r o u n d o f k n o w l e d g e from m o d e l studies a n d theory.


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Simple Electron-Transfer Proteins H o w closely do these proteins resemble solid-state electron-transfer matrices w i t h w h i c h w e are familiar, such as h o p semiconductors? I n m y o w n m o d e l w o r k (I) o n electronic c o n d u c t i o n i n what is effectively a s o l i d , crystal or glass, matrix, w e made various m i x e d - v a l e n t solids f r o m c o m p l e x ions a n d analyzed t h e i r c o n d u c t i v i t y (Table II). S o m e w e r e q u i t e good conductors, b u t the o v e r w h e l m i n g i m p r e s s i o n this research made u p o n m e was that the cooperative electrostatics of the lattice made it difficult to create r a p i d charge m o v e m e n t b y a h o p m e c h a n i s m i n a m o l e c u l a r solid (I).


Table II. Some Examples of M o d e l Semiconductor Crystals Compound

Log (Resistivity), 300 °C 6

+

5

3

4

3

2

4

6

3

3

2+

6

Activation Energy, ev

~8 -10 6 >8 -2 10

KFeFe(CN) M (CuCl ) -(CuCl ) Tl Fe(CN) [Fe(o-phen) ] [IrCl ] Fe 0 Crocidolite" 2

4

0.75 0.3 0.4 >1.0 0.1 >0.5

NOTE: In these solids, high conductivity and low activation energy were observed only for very short metal-to-metal distances. Fe(II), Fe (III) asbestos. fl

I w o u l d suggest that those w h o study s i m p l e electron-transfer chains s h o u l d look at the p r o b l e m s w e met. W e also investigated m a n y black i n organic i o n i c lattice solids at r o o m t e m p e r a t u r e , b u t failed to find any r e a l l y good conductors ( u n p u b l i s h e d observations). I was astonished w h e n others f o u n d superconductors i n some of these families. A great advantage of a p r o t e i n matrix is that the electrostatics are not cooperative, as is obviously the case i n an N a C l matrix. M o r e o v e r , a l t h o u g h crystal lattices are r i g i d a n d proteins crystallize i n o r d e r e d matrices, p r o t e i n molecules are not crystallites. T h e y m o r e closely r e s e m b l e glasses o r e v e n r u b b e r l i k e molecules that are easily open to l o w - e n e r g y m i n o r relaxations. T y p i c a l examples are the i r o n - s u l f u r proteins d e s c r i b e d i n T a b l e I I I . H o w ever, t h e i r relaxation is also l i m i t e d b y c r o s s - l i n k i n g , as i n a h a r d r u b b e r . T h e F e S proteins are c r o s s - l i n k e d b y the F e - S l i n k s , b u t they are also b u i l t of P sheets c r o s s - l i n k e d over remote parts of the sequence. T h e y are n

n

Table III. Structural Features of F e - S Proteins Protein

Structural Feature

Ferredoxin (Fe S ) Ferredoxin (Fe S ) HIPIP* (Fe S ) Rubredoxin (Fe) 4

2

2

4

4

4

Antiparallel p sheet Short antiparallel sheet Three-stranded P sheet Small sheet segments

"HIPIP is high-potential iron protein.


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9

found i n fixed positions i n biological organization. F i n a l l y , the e l e c t r o n transfer centers are b u r i e d away from solvent, so the charge s w i t c h does not i n v o l v e solvent electrostatic energies. It is w o r t h stressing some other points from Table I I . I n m i x e d - v a l e n t crystals, w e found strong i n t e r - c o m p l e x - i o n charge-transfer spectral absorp tion bands o n l y at v e r y short distances (e.g., F e 0 a n d P r u s s i a n blue). A t somewhat longer distances (e.g., the C u ( I ) - C u ( I I ) system) the bands w e r e m u c h w e a k e r a n d w e r e not detected i n the [ F e ( o - p h e n ) ] [ I r C l ] ~ systems, w h e r e o-phen is 1,10-orthophenanthroline. T h e y have not b e e n detected i n complexes of s i m p l e electron-transfer proteins. T h e overlap i n t e g r a l i n these systems m u s t be v e r y s m a l l i n d e e d . 3

4


3

2+

6

2

C o n s i d e r a m e t a l center taken from such a p r o t e i n , b u t n o w s u r r o u n d e d b y a p r o t e i n sphere of radius close to the shortest distance to the p r o t e i n surface, for example, 10 A . I n this p o s i t i o n it can m a k e the usual p r o t e i n - p r o t e i n contact distance for biological e l e c t r o n transfer i n a l l directions. A crystal from an e q u i m o l a r m i x t u r e of such molecules i n its two oxidation states w i l l show an e x t r e m e l y weak charge-transfer b a n d a n d it w i l l be a v e r y p o o r conductor. T h i s characteristic is generated not because of the rather l o w n u m b e r of potential carriers, n o r because of a h i g h activation energy, b u t because the overlap is so s m a l l i n the lattice a n d m o t i o n is restricted. C o n t r a s t this situation to a cluster, F e S , w i t h i n w h i c h the c o n d u c t i v i t y w i l l be h i g h , as i n F e 0 (Table II). B i o l o g i c a l systems can m a k e F e 0 or F e O ( O H ) crystals, b u t do not use t h e m i n electronic devices. B i o l o g i c a l e l e c t r o n transfer is slow, e v e n i n proteins w h e r e some or a l l of the barriers f o u n d i n crystals have b e e n r e m o v e d to gain v e r y selective local c i r c u i t pathways. n

3

n

4

3

4

Analysis. S h o r t l y after o u r " f a i l u r e s " to d e v e l o p suitable electronic c o n d u c t i v i t y devices i n m i x e d - v a l e n t crystals, w e d e v o t e d our attention to the analysis of what s i m p l e electron-transfer proteins are r e a l l y l i k e . W e a n d others n o w have v e r y detailed N M R spectroscopic studies of two series: cytochromes c a n d b (2, 3, 15) a n d b l u e c o p p e r proteins (4, 5). T h e s e are all s i m p l e electron-transfer proteins. 5

I shall concentrate o n the properties of c y t o c h r o m e c. W e have full (90%) p r o t o n N M R spectroscopic assignments for yeast, t u n a , a n d horse cyto c h r o m e c i n b o t h Fe(II) a n d Fe(III) states as w e l l as 3 crystal structures for all the proteins (6-8). T h e N M R spectroscopic w o r k established not j u s t the resemblance of the solution a n d crystal structures, b u t the m a n y d y n a m i c features of these proteins. Space does not p e r m i t d e t a i l e d analysis h e r e , a n d I shall therefore state the conclusions b r i e f l y . T h e r e is a general b e l i e f that proteins are to be c o m p a r e d w i t h o r d e r e d crystal solids. O u r N M R spectroscopic data a c c u m u l a t e d since 1972 do not m a t c h this p i c t u r e , w h i c h w e believe to have arisen t h r o u g h a m i s i n t e r p r e -


10


tation of X - r a y diffraction data. T h e X - r a y data, diffraction patterns, reflect the h i g h l y p o p u l a t e d states of proteins i n a regular lattice s u r r o u n d e d b y water. T h i s water contributes little to the diffraction p a t t e r n because m u c h of it is i n a l i q u i d state. O n l y a v e r y s m a l l n u m b e r of water m o l e c u l e s have residence times i n excess of 10~ s. 9

T h e outside of proteins (e.g., lysines) m u s t be i n r a p i d l y fluctuating states. T h i s r a p i d fluctuation is c o n f i r m e d b y the n a r r o w l i n e w i d t h s of €-CH

2

groups of lysines i n N M R spectra. T h e i n s i d e of proteins has b e e n

represented as fixed to a fixed secondary structure. T h e degree to w h i c h this structure fluctuates can b e d e t e r m i n e d i n part e x p e r i m e n t a l l y b y u s i n g N M R spectroscopic analysis of, for example, aromatic r i n g flip rates, N i f - N D Downloaded by 185.13.32.143 on July 5, 2016 | http://pubs.acs.org Publication Date: May 5, 1989 | doi: 10.1021/ba-1990-0226.ch001

exchange, a n d relaxation times, especially for isolated

1 3

C n u c l e i . T h e results

of s u c h studies give a m o b i l i t y m a p of a p r o t e i n ( F i g u r e 3) (2).

Figure 3. A mobility map imposed on the structure of cytochrome c. Some fast-moving flipping or flapping groups are hatched. Some slow-moving groups are filled in. Helices are illustrated. (Reproduced from ref. 2.)


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WILLIAMS

A m o r e d e t a i l e d m a p s h o w i n g l i n e - w i d t h studies t h r o u g h o u t a p r o t e i n w o u l d d i s t i n g u i s h atoms that have N M R relaxation times e q u a l to t h e t u m b l i n g t i m e o f the p r o t e i n , approximately 1 0 s" for a 10,000-dalton p r o t e i n , from those that t u m b l e m o r e r a p i d l y . A l l parts o f the major secondary s t r u c t u r e are resistant to v e r y fast i n d e p e n d e n t m o t i o n . O n a l o n g e r t i m e scale, slower motions o f considerable a m p l i t u d e affect the intensities of cross-peaks i n t w o - d i m e n s i o n a l N M R spectra. F u r t h e r m o r e , signal averaging is possible w h e n m o r e than one site is o c c u p i e d b y a g i v e n atom for appreciable lengths of t i m e . T h e s e analyses show that motions o f the m a i n c h a i n increase i n loops o f t h e structure relative to helices a n d (3 sheets. T h e y also show that relative motions o f m a i n - c h a i n segments m u s t exceed 1.0 A o n t h e t i m e scale o f 1 0 s" . T h i s conclusion follows from t h e observation o f aromatic r i n g flipping w i t h this rate constant inside most p r o t e i n s , b u t noticeably not i n c y t o c h r o m e c. 9

5


11


1

1

Molecular Dynamics. Since proteins w e r e first d e s c r i b e d i n these t e r m s , there has b e e n a v e r y considerable advance i n c o m p u t a t i o n a l studies of m o l e c u l a r m o t i o n i n proteins. T h e s e m o l e c u l a r d y n a m i c calculations give a p i c t u r e o f a p r o t e i n as a h i g h l y fluctuating assembly. F r o m b o t h this w o r k w i t h the e x p e r i m e n t a l N M R spectroscopic data a n d today's X - r a y diffraction B factors, w e c a n b e certain that a m i n o a c i d side chains such as valines a n d leucines flip as r a p i d l y as phenylalanines. H o w e v e r , t h e y are a s y m m e t r i c tops a n d the m o t i o n is h a r d to detect. A major c o n c e r n , t h e n , is not j u s t t h e structure o f a p r o t e i n a n d its relationship to f u n c t i o n , b u t the w a y i n w h i c h the d y n a m i c s w i t h i n t h e structure u n d e r l i e s function (9, 10). E l e c t r o n - t r a n s f e r proteins are a p a r t i c u l a r l y good case for study. So far electron-transfer proteins have b e e n d e s c r i b e d m a i n l y i n terms o f isotropic m o t i o n . T h e larger-scale motions of a h e l i x o r of a segment causing a groove-opening reaction are anisotropic, slow (>10~ s), a n d difficult to detect except b y p e r t u r b a t i o n o f the w h o l e struc t u r e . H o w e v e r , s u c h slow large-scale motions as these are r e q u i r e d to e x p l a i n some reactions a n d m u c h N H - N D exchange i n proteins. T h e y w i l l b e seen to b e o f great functional consequence later. 4

Stiffness. T h e s i m p l e electron-transfer p r o t e i n , c y t o c h r o m e c, a l t h o u g h i t is a h e l i c a l p r o t e i n , is one o f the stiffest proteins w e have e x a m i n e d ( F i g u r e 3). It is c r o s s - l i n k e d . T h e b l u e c o p p e r proteins are f o r m e d f r o m a stiff (3-sheet b a r r e l , a n d cytochrome b is s u p p o r t e d b y a (3 sheet (see F i g u r e 2). (3 sheets are stiff because they are c r o s s - l i n k e d b y H bonds. I n t h e b l u e p r o t e i n s , the c o p p e r changes valence state o r c a n b e r e m o v e d w i t h l i t t l e effect o n the structure. T h i s c o n d i t i o n is t h e basis o f the entatic state h y pothesis ( J I ) . T h e o v e r a l l i m p r e s s i o n o f these proteins a n d o f t h e F e S proteins (again (3 sheets, F i g u r e 1) is that t h e uptake o r loss o f a n e l e c t r o n has little effect o n the p r o t e i n conformation. T h i s lack o f effect confirms t h e i r 5

n


n

12


d e s c r i p t i o n as s i m p l e electron-transfer proteins. T h e i r stiffness is not to be confused w i t h the r i g i d i t y o f crystals. T h e i n t e r i o r o f a l l these proteins r e mains q u i t e m o b i l e o n the t i m e scale of the electron-transfer reactions.


F r o m other studies w e k n o w that other h e l i c a l proteins that are not c r o s s - l i n k e d are r e a d i l y adjustable b y i o n b i n d i n g (calmodulin) (10), change o f oxidation state or oxygenation ( h e m o g l o b i n , h e m e r y t h r i n , h e m o c y a n i n ) , or b y p h o s p h o r y l a t i o n (phosphorylase) (12). W e m u s t look at a l l the e l e c t r o n transfer proteins i n terms o f t h e i r secondary structure a n d the c r o s s - l i n k i n g of that structure. Before t u r n i n g to m o r e m o b i l e frameworks, w e n e e d to inspect i n m o r e d e t a i l the significance o f i n d i v i d u a l a m i n o acids i n the se quences of the s i m p l e electron-transfer p r o t e i n s , b o t h i n the i n t e r i o r a n d o n the p e r i p h e r y . Interior Side C h a i n s a n d E l e c t r o n - T r a n s f e r Rates. T h e r e is the suggestion that, i n d e p e n d e n t of the redox p o t e n t i a l of the metals i n e l e c t r o n transfer proteins, aromatic groups i n the i n t e r i o r of proteins c a n assist e l e c t r o n transfer. T h i s suggestion is contrary to o u r experience i n two respects. F i r s t , i f i t w e r e t r u e , e l e c t r o n transfer from the h e m e of c y t o c h r o m e c c o u l d p r o c e e d t h r o u g h t r y p t o p h a n - 5 9 to the w r o n g side o f the p r o t e i n . T h i s result is not observed. S e c o n d , there is no e v i d e n c e from models that e l e c t r o n transfer rates from centers that have redox potentials of a r o u n d + 0 . 2 V are affected b y aromatic residues outside the c o o r d i n a t i o n sphere (13). H o w e v e r , i n v i e w o f the assertion that aromatic residues c o u l d have s u c h an effect, w e e x a m i n e d the electron-transfer rates i n the f o l l o w i n g two u n i m o l e c u l a r r e actions. 1. E l e c t r o n exchange b e t w e e n two cytochromes c, self-exchange, w h e r e the two proteins are h e l d together b y hexametaphosphate anions ( H M P ) i n a c o m p l e x [ C y t . c . H M P ]

2

(14).

2. E l e c t r o n transfer b e t w e e n c y t o c h r o m e c a n d c y t o c h r o m e i n t h e i r c o m p l e x (14).

b

5

B o t h reactions can be e x a m i n e d b y N M R spectroscopic m e a s u r e m e n t s of e l e c t r o n exchange seen t h r o u g h l i n e - b r o a d e n i n g . T h e test of the effect o f aromatic groups has b e e n m a d e b y site-specific mutagenesis w h e r e P h e - 8 2 of cytochrome c (see F i g u r e 3) is r e p l a c e d b y T y r - 8 2 , G l y - 8 2 , or Ser-82 (6). T h e results are s h o w n i n T a b l e IV. N o assistance to electron-transfer rates of P h e - 8 2 a n d T y r - 8 2 relative to G l y - 8 2 or Ser-82 was f o u n d . W e m a i n t a i n the p o s i t i o n that aromatic a m i n o a c i d residues can assist e l e c t r o n transfer o n l y w h e r e the redox centers are at > + 0.8 V , s u c h as i n reactions of F e O ( I V ) states of peroxidases (13, 16). A second c l a i m (17) has b e e n made for the f u n c t i o n i n g of P h e - 8 2 i n the electron-transfer reactions of c y t o c h r o m e c w i t h c y t o c h r o m e b . A c c o r d i n g to this c l a i m , P h e - 8 2 is so m o b i l e that it can flap out into the space b e t w e e n 5


1.

WILLIAMS


13

Table IV. Electron-Transfer Rates of Cytochrome c Measured at Equilibrium by N M R Spectroscopy Self-Exchange Self-Exchange [Fe Cc, Fe Cb ] Intracomplex Bimolecular I s' Cytochrome c Variant [Fe Cc, Fe"Cb ], s' ll

!Il

5

a

c

b

lu

Horse: wild-type Yeast: C102T mutant Yeast: C102T, F82G mutant*

5

0.6 ± 0.3 ± 0.5 ±

rf

1800 ± 300 ± 1200 ±

200 100 100

400 ± 200 ± 400 ±

100 100 100

NOTE: Ref. 14. Cc is cytochrome c, and Cb is cytochrome b. "Determined by saturation transfer to Met-80 methyl resonance of Fe"Cc. Intensity decrease was independent of the F e C c / F e C c ratio and total protein concentration (10 raM phosphate, pH 7.1, 30 °C). Determined by saturation transfer to Met-80 methyl resonance of F e C c (100 mM phosphate, pH 7.0, 30 °C). determined from line broadenings: observed in the presence of hexametaphosphate (HMP), a polyphosphate anion known to cause aggregation of cytochrome c (pH 7.0, 30 °C). The intramolecular complex is [(Cyt c) (HMP) ]. The yeast C102T, F82Y behaves very like the native protein. The code C102T means that, in this yeast, cytochrome c residue cysteine (C) at position 102 of the sequence has been replaced by a threonine (T) residue. F82G means that phenylalanine (F) 82 in the sequence has been replaced by glycine (G). Y is tyrosine and S is serine. T h e yeast C102T,F82S behaves very like F82G. ni

H

b


0.2 0.1 0.2

1

1

n

2

2

d

the t w o cytochromes i n t h e i r complexes. O u r f u l l assignments of the two cytochromes separately a n d i n t h e i r c o m p l e x a l l o w us to state that no such m o v e m e n t of P h e - 8 2 is detectable. A l t h o u g h i t flips r a p i d l y i n the free cytochrome a n d i n the complex, there is no e v i d e n c e for flapping o u t w a r d at rates a r o u n d 1 0 s" (18). 5

1

T h e i n t e r i o r of a l l these proteins allows q u i t e considerable v i b r o n i c rotational m o t i o n . O u r e v i d e n c e indicates that m a n y aromatic rings i n most proteins flip r a p i d l y , > 1 0 s" . W e can observe few o t h e r motions i n the i n t e r i o r . T h e cavity size n e e d e d to flip P h e a n d T y r residues is such that w e b e l i e v e that m a n y V a l , L e u , H e , a n d M e t residues can rotate easily, a l t h o u g h t h r o u g h t h e i r a s y m m e t r y t h e y are seen i n one h e a v i l y p o p u l a t e d state (as i f they w e r e rigid). T h e exterior of the p r o t e i n a n d its motions b e c o m e p a r t i c u l a r l y i m p o r t a n t w h e n w e t u r n to the scavenger p r o t e i n s . 4

1

Surfaces o f Scavenger Proteins. T h e d i s t i n c t i o n b e t w e e n a scav enger p r o t e i n a n d a p r o t e i n that is a p e r m a n e n t part o f an organization lies not i n basic electron-transfer reactions, b u t i n the organization o f the p r o teins. Just as some classes of proteins have v e r y m o b i l e i n t e r i o r s a n d others have relatively r i g i d frameworks, so some proteins f o r m r e l a t i v e l y r i g i d as semblies a n d others d o not. Scavenger proteins (e.g., c y t o c h r o m e c) are often n e a r l y s p h e r i c a l a n d have v i r t u a l l y no concave surfaces. T h e scavenger proteins also have h i g h l y charged surfaces. T h e charges o n p r o t e i n surfaces attributable to G l u , A s p , L y s , a n d A r g side chains are k n o w n to b e r e l a t i v e l y m o b i l e themselves. F u r t h e r m o r e , t h e i r long-range electrostatic interactions w i t h other proteins allow considerable m o v e m e n t i n the complexes w i t h o u t


14


m u c h loss of energy. W i d e areas of the surface of c y t o c h r o m e c are zones of approximately the same electrostatic p o t e n t i a l . W e have e x a m i n e d these surfaces b y u s i n g c o m p l e x i o n probes. W e cannot define a b i n d i n g site for ions s u c h as [ C ^ C N ^ ] , b u t o n l y a b i n d i n g zone ( F i g u r e 4) (19). 3 -

T h e scavenger p r o t e i n has to b e able to find an electron-transfer site, react, a n d leave that site i n ~10 s to m a i n t a i n e l e c t r o n flow. Its b i n d i n g has to b e effectively diflusion-eontrolled to a l l o w a b i n d i n g constant o f 1 0 T h e easiest w a y to achieve such a reaction b e t w e e n surfaces is to have m a n y electrostatic m o b i l e fingers (lysines) that w i l l b i n d to m a n y electrostatic m o b i l e anions. T h e assembly is m o b i l e . W e call this h a n d - i n - g l o v e fitting. 3


6

o

T h e exact structure for the electron-transfer act m a y b e of l i t t l e c o n sequence. T h e surface d y n a m i c s is of greater i m p o r t a n c e . I n contrast, s o m e p r o t e i n s , such as m a n y F e „ S electron-transfer p r o t e i n s , are fixed parts o f organizations. W e suspect that the interactions they f o r m are based m o r e largely o n shorter range h y d r o p h o b i c forces than o n electrostatic forces. A g a i n , these proteins have m u c h m o r e d i s t i n c t i v e shapes, insofar as t h e y are k n o w n ( F i g u r e 2). It m a y w e l l b e that, w h e n b o u n d i n an o r g a n i z e d system, t h e i r surfaces are not m o b i l e . n

Simple Oxidases and Their Electron-Transfer Reactions S o m e o f the major s i m p l e oxidases are g i v e n i n T a b l e V . I n t h e i r reactions, oxidation of substrate is not c o u p l e d to any o t h e r reaction. F o r e x a m p l e , dioxygen + substrate - » o x i d i z e d p r o d u c t + r e d u c e d oxygen T h e reaction is w r i t t e n i n l o n g - h a n d because r e d u c e d oxygen can appear as 0 ~ , H 0 , o r H 0 . T h e alternative of O ^ i n c o r p o r a t i o n i n a n oxidation does not r e q u i r e electron transfer except i n i n n e r - s p h e r e activation (e.g., i n d i oxygenases of soil bacteria). 2

2

2

2

I n the case of plant peroxidases that oxidize phenols a n d i n d o l e s , the reaction is a one-electron oxidation b y peroxide. T h e i r o n cycles from Fe(III) to a n o m i n a l F e ( V ) (i.e., F e ( I V ) plus a free r a d i c a l , t h r o u g h F e O ( I V ) ) . T h e substrate goes to a free radical that m a y p o l y m e r i z e . W e s t u d i e d these electron-transfer proteins b y u s i n g N M R spectroscopy to d e t e r m i n e the structure w i t h substrate b o u n d . W e s h o w e d that e l e c t r o n transfer took place o v e r a distance of some 10 A (outer-sphere reaction) (20). O u r present interest lies i n the structure of these proteins. A l l the peroxidases i n this class of k n o w n structure are s i m i l a r to s i m p l e electron-transfer p r o t e i n s , i n that t h e y are c r o s s - l i n k e d e i t h e r c h e m i c a l l y (horseradish peroxidases) or b y (3 sheets (cytochrome c peroxidases). T h e pocket of the h i g h - s p i n h e m e is o p e n to small molecules, 0 or H 0 , b u t not to the larger substrates s u c h as p h e n o l s or indoles. E l e c t r o n transfer is not l i n k e d to a conformational change. O t h e r oxidases i n this class may w e l l i n c l u d e the c o p p e r oxidases s u c h as ascorbic 2

2

2


1.

WILLIAMS


15

ρ

•S

ο OÙ

α


« ο

|3 g §

si g &

Ο

'S?

ο «

•S έ

•S . 9 s S

•S ^

1 s

δ.


16


acid a n d p h e n o l oxidases, b u t w e do not k n o w the structure of the d i o x y g e n sites i n v o l v i n g t y p e 2 C u a n d the c o p p e r pairs of these proteins. W e have

Table V. Long-Range Electron Transfer to Aromatic Compounds Protein

Group Involved Possibly tryptophan Phenols or indoles Phenols Tyrosine(P), phaeophytin Tyrosine


Cytochrome c peroxidase Horseradish peroxidase Copper oxidases (laccase) Reaction center Ribonucleotide reductase

NOTE: All systems involve high redox potential centers. Quinones and flavins are, of course, very different aromatic electron-transfer centers from metal ions with low-lying molecular orbitals (redox potentials).

(a) Figure 5. The change in structure from (a) Fe(III) cytochrome c to (b) Fe(III)-CN~ cytochrome c. Johnson et al.; Electron Transfer in Biology and the Solid State Advances in Chemistry; American Chemical Society: Washington, DC, 1989.

1.

WILLIAMS


17

N M R spectroscopic e v i d e n c e that the inside of m a n y of these proteins is as m o b i l e as that of the s i m p l e electron-transfer proteins.

Conformational Changes: Groove Openings I n the d e s c r i p t i o n of c y t o c h r o m e c, w e have d e l i b e r a t e l y left aside one m u c h s t u d i e d reaction, that w i t h cyanide (21). T h e reaction is v e r y slow a n d q u i t e u n r e l a t e d to any fast, s i m p l e , electron-transfer reaction. H o w e v e r , it is suit able as a m o d e l for c o u p l e d reactions. A t temperatures above 60 °C, the electron-transfer reaction of c y t o c h r o m e c is c o u p l e d to a b r e a k i n g o f the M e t - 8 0 - F e ( I I I ) b o n d . W e shall consider the P450 reactions i n s i m i l a r t e r m s .


A d e t a i l e d N M R spectroscopic study w i t h f u l l assignment of the c y a n i d e Fe(III) state of the p r o t e i n (21) shows that the groove o p e n i n g is a l e v e r l i k e action i n w h i c h the segment of the c y t o c h r o m e b e l o w the s e q u e n t i a l c h a i n 79 to 85, w h i c h i n c l u d e s M e t - 8 0 , moves away from the h e m e to leave r o o m for the cyanide a n i o n ( F i g u r e 5). T h e reaction shows that r e l a t i v e l y s m a l l

(b) Figure 5.—Continued. Johnson et al.; Electron Transfer in Biology and the Solid State Advances in Chemistry; American Chemical Society: Washington, DC, 1989.

18


domains of a p r o t e i n can m o v e away from the b u l k , e v e n o n change

from

l o w - s p i n Fe(II) to h i g h - s p i n Fe(III). T h e reaction is l o w - s p i n Fe(II) - » l o w - s p i n Fe(III) —> h i g h - s p i n Fe(III) —» c y a n i d e c o m p l e x W e shall next look for s i m i l a r changes i n other p r o t e i n s , n o t i n g that i n c y t o c h r o m e c the m o v a b l e part of the p r o t e i n is not c r o s s - l i n k e d .

Coupled Electron Transfer T h e s e are the most difficult proteins to study. A m o n g i r o n p r o t e i n s , t h e y i n c l u d e h e m o g l o b i n , w h i c h is l i n k e d to a c y t o c h r o m e b r e d u c t i o n to protect it; h e m e r y t h r i n ; c y t o c h r o m e P , w h i c h is l i n k e d to e i t h e r a c y t o c h r o m e b or an F e S p r o t e i n to ensure the c o n t r o l l e d o r d e r of its reaction k i n e t i c s ; a n d cytochromes b a n d a o f the electron-transfer chains of chloroplasts a n d m i t o c h o n d r i a , i n w h i c h e l e c t r o n transfer is c o u p l e d to p r o t o n m o v e m e n t . A m o n g c o p p e r proteins, t h e y i n c l u d e the dioxygen c a r r i e r h e m o c y a n i n a n d the c y t o c h r o m e oxidase c e n t e r , C u .


5

4 5 0

n

5

n

B

C o u p l i n g h e r e is not a c o u p l i n g of the electrostatic potentials of the electron-transfer ions w i t h electrostatic potentials o f o t h e r groups, as i t m a y b e i n c y t o c h r o m e c . C o u p l i n g is k n o w n to i n v o l v e conformational changes (22-25). T h i s result is s h o w n not o n l y t h r o u g h the properties of the p r o t e i n s , b u t b y f o l l o w i n g the properties of the m e t a l i o n (for e x a m p l e , its s p i n state o r e l e c t r o n paramagnetic resonance ( E P R ) signals). 3

T h e i n f o r m a t i o n o b t a i n e d about the m e t a l centers themselves is r e v e a l i n g . T h e c o o r d i n a t i o n sphere of the c o p p e r , as seen d i r e c t l y b y X - r a y structure d e t e r m i n a t i o n o r b y various p h y s i c a l m e a s u r e m e n t s , is u n l i k e that f o u n d i n the b l u e c o p p e r proteins d e s c r i b e d as s i m p l e electron-transfer proteins. It is l i k e l y that there are no m e t h i o n i n e or cysteine ligands, b u t o n l y h i s t i d i n e s . A g a i n , the copper(II) E P R signals are m o r e closely those of d i s t o r t e d tetragonal Cu(II) sites i n m o d e l s . I n the case of the h e m e proteins i n this class, the indications are that the i r o n changes e i t h e r s p i n state o r the l i g a n d geometry a r o u n d i t ( E P R data) d u r i n g reaction. T h e indications are that the m e t a l i o n sites themselves differ f r o m those of the s i m p l e elec tron-transfer h e m e proteins. H o w e v e r , they d o not differ greatly from some of the sites of the s i m p l e oxidases. T h e differences i n f u n c t i o n are r e l a t e d to the differences i n p r o t e i n fold a n d its m o b i l i t y . T h e simplest case to consider first is that of h e m o g l o b i n . I n h e m o g l o b i n the dioxygen uptake reaction of h i g h - s p i n Fe(II) o r the redox s w i t c h of h i g h s p i n Fe(II) to h i g h - s p i n Fe(III) h y d r a t e is a c c o m p a n i e d b y a considerable conformational change ( F i g u r e 6). T h e change involves some m o v e m e n t o f the i r o n atom t o w a r d the h e m e p l a n e , some adjustment of the i r o n ligands, b o t h the h i s t i d i n e a n d the p o r p h y r i n , a n d most i m p o r t a n t l y , the adjustment of helices. T h e adjustment of helices is, w e consider, a c o m m o n feature of


1.

WILLIAMS


19

all these c o u p l i n g proteins. T h e e v i d e n c e that c o u p l i n g is r e l a t e d to h e l i x m o v e m e n t s comes f r o m general studies of proteins other t h a n h e m o g l o b i n (e.g., the c a l c i u m trigger proteins a n d phosphorylase). C o u p l i n g is also v e r y l i k e l y i n the cooperative action o f h e l i c a l h e m e r y t h r i n a n d h e m o c y a n i n , w h e r e the i r o n a n d c o p p e r ions u n d e r g o redox switches o n d i o x y g e n uptake rather t h a n spin-state changes.


"Fe-

HIGH SPIN OR Fe(O)

LOW SPIN OR Fe(IO)

Figure 6. A representation of coupled helix movements with chemical changes at the iron atom of a heme protein.

A r e v e a l i n g example is p r o v i d e d b y o u r study of c y t o c h r o m e b ^ . T h e structure, s h o w n i n F i g u r e 7, is a four-helix b u n d l e p r o t e i n w i t h the i r o n b o u n d as i n c y t o c h r o m e c (26). T h e redox p o t e n t i a l o f the p r o t e i n is p H d e p e n d e n t ; there is a s w i t c h of 260 m V w i t h i n the p H range 5 . 0 - 8 . 5 . T h i s p r o t o n - c o u p l e d redox p o t e n t i a l lies i n the p h y s i o l o g i c a l p H range. W e k n o w that a conformational change accompanies the p r o t o n a t i o n - r e d o x s w i t c h . I n this respect, the p r o t e i n is e n t i r e l y comparable w i t h h e m o g l o b i n , b u t the reactions are n o w c o n f i n e d to l o w - s p i n states. T h e s e reactions are t o b e contrasted w i t h those o f c y t o c h r o m e c, F e „ S , a n d the b l u e c o p p e r p r o t e i n s d e s c r i b e d i n the section o n s i m p l e electron-transfer p r o t e i n s . G i v e n the obvious ease of h e l i x - h e l i x m o t i o n s ; the strong e v i d e n c e for the h i g h l y h e l i c a l character of the m e m b r a n e portions of c y t o c h r o m e oxidase (i.e., c o n t a i n i n g c y t o c h r o m e a a n d C u ) , of the c y t o c h r o m e b c o m p l e x , a n d of c y t o c h r o m e P ; a n d the fact that these h e m e proteins are not crossl i n k e d b y e i t h e r e x t e n d e d (3 sheets or c h e m i c a l cross-links, i t appears that c o u p l i n g i n a l l these cases is a p r o p e r t y of h e l i x m o t i o n (20). T h i s is not the place to e x t e n d the a r g u m e n t , b u t a w h o l e range o f o t h e r c o u p l e d activities of proteins appears to b e associated w i t h h e l i x m o t i o n (27). It is i m m e d i a t e l y clear b y c o m p a r i s o n w i t h s i m p l e electron-transfer proteins that c r o s s - l i n k i n g separates the p r o t e i n structures o f c o u p l e d a n d u n c o u p l e d activities (27). n

3

B

4 5 0



20


Figure 7. The structure of cytochrome bm (opposite page) showing the groups likely to be responsible for the pH-dependent redox potential (see ref 26). On this page is shown the binding mode of two molecules in the crystals and the hydrogen-bonding network. (Reproduced from ref 26.) A t present w e can describe the motions o f the helices o n l y b y analogy w i t h c a l m o d u l i n a n d h e m o g l o b i n . H e r e is one major task for the c h e m i s t i n t e r e s t e d i n biological e l e c t r o n transfer.

Other Electron-Transfer Centers T h e concentration o n b l u e c o p p e r centers a n d h e m e i n electron-transfer proteins has t e n d e d to h i d e the d i v e r s i t y o f such centers. E a r l i e r reference was m a d e to F e S centers as t y p i c a l of u n c o u p l e d almost-solid-state devices. O t h e r centers are s i m i l a r , such as N i - S centers o f d i h y d r o g e n reactions a n d c o b a l a m i n centers of m e t h a n e c h e m i s t r y . (The v i t a m i n B chemistry i n rearrangement e n z y m e s seems to d e p e n d o n h o m o l y t i c fission g i v i n g free radicals a n d not o n electron transfer; it is based o n atom m o v e m e n t s . ) Yet o t h e r centers i n c l u d e F e - O - F e (or M n - O - M n ) for g e n e r a t i n g tyrosine-free radicals i n r i b o n u c l e o t i d e reductase; [ M n O ] i n the d i o x y g e n - l i b e r a t i n g c e n n

n

1 2

n



1.

21


WILLIAMS

Figure 7.—Continued

ter o f photosystem I I ; type 2 a n d t y p e 3 c o p p e r i n c o p p e r oxidases; m o l y b d e n u m i n the t w o - e l e c t r o n reactions of N 0 ~ , S 0 ~ , a n d a l d e h y d e s ; a n d a 3

4

2

considerable n u m b e r of s i m p l e i r o n e n z y m e s that m a y w e l l use i n n e r - or o u t e r - s p h e r e electron-transfer m e c h a n i s m s (e.g., the oxidative e n z y m e s o f secondary metabolism). T h e most i n t r i g u i n g centers c o n t a i n t w o o r m o r e m e t a l ions a n d partake i n reactions r e l a t e d to d i o x y g e n r e d u c t i o n or release. T h e e q u i v a l e n t e l e c t r o n i c states are M ( I I ) 0 * M ( I I ) , M ( I I I ) - 0 « M ( I I I ) , M ( I V ) 0 * 0 M ( I V ) , 2

2

2

2

a n d the various m i x e d - v a l e n t i n t e r m e d i a t e s for the d i o x y g e n T h e r e are also c o r r e s p o n d i n g r e d u c e d and M ( I I I ) 0 X ) ~ M ( I I I ) . However, 2

2

r e a c t i o n s u c h as M ( I I ) ( H 0 ) 2

2

we

states s u c h as

2

molecule.

M(II)0 "*M(II) 2

2

m u s t i n c l u d e states o f p r o t o n

M(II), M ( I I I ) ( O H X O H - ) M ( I I I ) , and

M ( I V ) O H " ~ H O M ( I V ) . It appears as i f these changes m u s t b e l i n k e d to #

conformational switches a n d to m o v e m e n t s o f protons d e e p i n s i d e p r o t e i n s , such as c y t o c h r o m e oxidase. T h e s e series m a y contain a larger n u m b e r of m e t a l ions o r a n u n e v e n n u m b e r o f protons. C h o i c e of m e t a l i o n (i.e., c o p p e r , manganese, o r iron) a n d c h o i c e o f p r o t e i n f o l d for a g i v e n m e t a l i o n c a n p r o v i d e c o n t r o l o v e r t h e r m o d y n a m i c balance b e t w e e n oxygen species. A n u n s o l v e d p r o b l e m is


22


the r e s o l u t i o n of questions p o s e d b y these electron-transfer centers, s u c h as w h y this or that m e t a l or particular choice of ligands is best for a g i v e n function. W h a t is the nature of the p r o t e i n selected b y evolution? I have t r i e d to answer these types of questions for a l i m i t e d series of e l e c t r o n transfer proteins, b u t m a n y other p r o b l e m s i n the electron-transfer systems of b i o l o g y clearly r e m a i n u n t o u c h e d .

Acknowledgments


I thank G . R. M o o r e , G . W i l l i a m s , G . P i e l a k , D . C o n c a r , D . W h i t f o r d , Y. G a o , a n d N . V e i t c h for t h e i r v e r y considerable h e l p w i t h b o t h the e x p e r i m e n t a l w o r k a n d the discussion that l i e b e h i n d this o v e r v i e w .

References 1. Williams, R. J . P. In Non-heme Iron Proteins; San Pietro, A., Ed.; Antioch Press: Yellow Springs, OH, 1965; Chapter 1, pp 7-15. 2. Williams, R. J . P. Z. Phys. Chem. 1988, 269, 387-402 and references therein. 3. Whitford, D . ; Veitch, N . ; Concar, D . W.; Williams, R. J . P. FEBS Lett. 1988, 238, 49-55. 4. Driscoll, P. C.; Hill, H . A . O.; Redfield, C . Eur. J. Biochem. 1987, 170, 279. 5. King, G . C.; Wright, P. E . Biochemistry 1986, 25, 2364, and personally com -municated work. 6. Tanako, T.; Dickerson, R. E . J. Mol. Biol. 1981, 153, 95. 7. Colman, P. M.; Freeman, H . C.; Guss, J . M.; Murata, M.; Norris, V. A . ; Ramshaw, J . A . M.; Ventatappa, M. P. Nature 1978, 272, 319-324. 8. Adman, E . T.; Jensen, L . H. Isr. J. Chem. 1981, 21, 8. 9. Williams, G.; Moore, G . R.; Williams, R. J . P. Comments Inorg. Chem. 1985, 4, 55. 10. Williams, R. J . P. Carlsberg Res. Commun. 1987, 52, 1. 11. Vallee, B. L.; Williams, R. J . P. Proc. Natl. Acad. Sci. U.S.A. 1968, 59, 498. 12. Prang, S. R.; Archarya, K . R.; Goldsmith, E . J.; Stuart, D . I.; Varvill, K . ; Fletterick, R. J.; Madsen, N . B . ; Johnson, L . N. Nature 1988, 336, 215-221. 13. Burns, P. S.; Harrod, J. F.; Williams, R. J. P.; Wright, P. E.; Biochim. Biophys. Acta 1976, 428, 261. 14. Concar, D . ; Gao, Y.; Pielak, G.; Whitford, D . ; Williams, R. J . P., unpublished work. 15. Wand, A . J.; DiStefano, D . L.; Feng, Y.; Roder, H.; Englander, S. W. Bio-chemistry 1989, 28, 186-194 and 195-203. 16. Pielak, G . J.; Concar, D . W.; Moore, G . R.; Williams, R. J . P. Protein Eng. 1987, 1, 83. 17. Wendoloski, J. J.; Matthew, J. B . ; Weber, P. C.; Salemme, F. R. Science 1987, 238, 794. 18. Moore, G . R.; Veitch, N.; Williams, R. J . P., unpublished results. 19. Arean, C. O.; Moore, G . R.; Williams, G.; Williams, R. J . P. Eur. J. Biochem. 1988, 173, 607. 20. Burns, P. S.; Williams, R. J . P.; Wright, P. E . J. Chem. Soc., Chem. Commun. 1975, 338-339. 21. Gao, Y.; Williams, R. J . P., unpublished results.


1.

WILLIAMS


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22. Poulos, T. L.; Sheriff, S.; Howard, A . J . J. Biol. Chem. 1987, 272, 13881. 23. Poulos, T. L . ; Finzel, B. C.; Gunzalus, I. C.; Wagner, G . C.; Krant, J . J. Biol. Chem. 1985, 260, 16122. 24. Wikstrom, M. Chem. Scr. 1987, 27B, 53. 25. Williams, R. J . P. FEBS Lett. 1987, 226, I. 26. Moore, G . R.; Williams, R. J . P.; Peterson, J.; Thomson, A . J.; Mathews, F. S. Biochim. Biophys. Acta 1985, 829, 83. 27. Williams, R. J . P. In The Enzymes of Biological Membranes; Martonosi, A . N., E d . ; Plenum: New York, 1985; Vol. 4, pp 71-110. 28. Williams, R. J . P. Proc. Roy. Soc. London 1981, B213, 361-397. 29. Tsukihara, T.; Fukuyama, K . ; Nakamura, M.; Katsube, Y.; Tanaka, N.; Kakudo, M.; Wada, K . ; Mase, T.; Matsubara, M. J. Biochem. (Tokyo) 1981, 90, 17631773. RECEIVED for review May 1, 1989. A C C E P T E D revised manuscript August 15, 1989.


Electron Transfer in Biology and the Solid State - American Chemical

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