Electron Transfer in Inorganic, Organic, and Biological Systems

11 Effects of Reaction Free Energy Downloaded by NORTH CAROLINA STATE UNIV on November 17, 2012 | http://pubs.acs.org Publication Date: May 5, 1991 | doi: 10.1021/ba-1991-0228.ch011

in Biological Electron Transfer In Vitro and In Vivo George McLendon , David Hickey , Albert Berghuis , Fred Sherman , and Gary Braver 1

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Department of Chemistry and Department of Biochemistry, University of Rochester, Rochester, NY 14627 Department of Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1W5, Canada

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Although much work detailed in this volume and elsewhere has fo cused on the determinants of protein electron-transfer rates in vivo, far less is known about the control of electron transfer in vivo. To this end, we here report results concerning the in vitro and in vivo reactivity of cytochrome c and a single-site replacement, Ν521. This mutant, despite a significant (50 mV) change in redox potential, sur prisingly does not have a deleterious effect on growth in vivo. Thus, a possible mechanism is proposed for regulation of growth of yeast on lactate.

WORK

O N B I O L O G I C A L L Y R E L E V A N T E L E C T R O N T R A N S F E R over the last decade has transformed o u r u n d e r s t a n d i n g of the f u n d a m e n t a l c h e m i c a l aspects of this k e y b i o c h e m i c a l reaction. T h e o r e t i c a l advances b u i l t o n the p i o n e e r i n g w o r k of M a r c u s a n d others (1-3) have o u t l i n e d the effects o n rate of d o n o r - a c c e p t o r distance, reaction free energy, i n t e r n a l m o l e c u l a r m o t i o n a n d solvent m o t i o n , a n d solvent relaxation d y n a m i c s .

E x p e r i m e n t a l tests of each of these p r e d i c t i o n s have b e e n made t h r o u g h i n v i t r o e x p e r i m e n t s (4-8). F o r example, G r a y a n d co-workers (4, 5) have systematically v a r i e d the d o n o r - a c c e p t o r distance i n proteins b y c o m p l e x i n g redox-active R u adducts to specific histidines located o n the surface of p r o teins l i k e c y t o c h r o m e c, m y o g l o b i n , a n d a z u r i n . B y l o o k i n g at several sites, 0065-2393/91/0228-0179$06.00/0 © 1991 American Chemical Society

In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, J., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1991.

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E T IN INORGANIC, ORGANIC, A N D BIOLOGICAL SYSTEMS

t h e y f o u n d e x p o n e n t i a l d e p e n d e n c e o f rate (fc ) o n distance (R), k °c e x p ( - 0 . 9 R ) , i n accord w i t h b o t h t h e o r y a n d previous e x p e r i m e n t s o n s m a l l m o l e c u l e e l e c t r o n transfer.

Downloaded by NORTH CAROLINA STATE UNIV on November 17, 2012 | http://pubs.acs.org Publication Date: May 5, 1991 | doi: 10.1021/ba-1991-0228.ch011

et

et

I n c o m p l e m e n t a r y w o r k , o t h e r groups have e x a m i n e d e l e c t r o n transfer i n p h y s i o l o g i c a l p r o t e i n - p r o t e i n complexes (6-8). B y s u b s t i t u t i n g different m e t a l - c o n t a i n i n g p o r p h y r i n s into the active sites of proteins l i k e h e m o g l o b i 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 c peroxidase, the reaction free energy c o u l d b e c o n t i n u o u s l y v a r i e d . F r o m M a r c u s t h e o r y , the electron-transfer rate c o n stant d e p e n d s o n reaction free e n e r g y a n d reorganization energy, k °c exp(-AGVfcT) et

AG* =

(

A

G

°

4λ

X ) 2

w h e r e A G * is activation free energy, k is the B o l t z m a n n constant, Τ is absolute t e m p e r a t u r e , A G is reaction free energy, a n d λ is reorganization energy. A n example o f this d e p e n d e n c e for a biological p r o t e i n - p r o t e i n redox c o u p l e , the c y t o c h r o m e c - c y t o c h r o m e c peroxidase c o m p l e x , is s h o w n i n F i g u r e 1. A fit to the M a r c u s equation gives an estimate of λ — 1.4 eV, 0

10

h

Figure 1. Dependence of the rate constant for oxidation on reaction free energy (AG) for the system [Fe(IV)0] cytochrome c peroxidase-(M^cytochrome c [where M is Fe \ Zn , or (H )J. The solid line is a fit to the Marcus equation, with λ = 1.4 eV l

11

+


11.

M C L E N D O N ET AL.

Effects of Reaction Free Energy

181

w h i c h is corroborated b y a d i r e c t m e a s u r e m e n t of the activation free e n e r g y i n this system. S u c h studies suggested that i n p r o t e i n complexes e l e c t r o n transfer is a c c o m p a n i e d b y significant reorganization of the p r o t e i n s t r u c t u r e . I n M a r c u s ' t e r m s , the reorganization energy, λ, is large; λ ~ 0 . 8 - 1 . 5 V. T h e l i m i t e d data for such complexes also support a strong e x p o n e n t i a l d e p e n d e n c e of the electron-transfer rate o n the distance b e t w e e n the e l e c t r o n -


d o n o r group a n d the acceptor. A l t h o u g h s u c h studies have clarified the f u n d a m e n t a l parameters that are i m p o r t a n t i n c h e m i c a l l y a n d b i o c h e m i c a l l y relevant electron-transfer reactions, t h e y do not d i r e c t l y test to w h a t extent these p h y s i c a l parameters affect the net m e t a b o l i c electron-transfer flux (due to e l e c t r o n transport) i n v i v o . W e assume that because these parameters c o n t r o l rates u n d e r s i m p l e i n v i t r o laboratory conditions, t h e y w i l l exercise s i m i l a r c o n t r o l i n v i v o . I call this the first l a w of biophysics: " I f w e can observe s o m e t h i n g , it m u s t be i m p o r t a n t . " C l e a r l y , basic p h y s i c a l a n d c h e m i c a l p r i n c i p l e s operate e q u a l l y i n s i d e a n d outside l i v i n g cells. It is less clear h o w sensitive the net m e t a b o l i c fluxes ( w h i c h are the k e y to c e l l u l a r growth) are to s m a l l changes i n the reaction free energy. W e have therefore chosen to examine d i r e c t l y h o w changes i n b i o l o g i c a l redox p o t e n t i a l affect i n v i v o m e t a b o l i s m . Specifically, the reaction free e n e r g y of the k e y m e t a b o l i c electron-transfer steps i n v o l v i n g c y t o c h r o m e c have b e e n c h a n g e d b y c h a n g i n g the redox p o t e n t i a l of c y t o c h r o m e c w i t h s i t e - d i r e c t e d mutagenesis. T h e s e inorganic-based redox reactions offer a u n i q u e o p p o r t u n i t y to explore free-energy effects o n m e t a b o l i s m . F o r m e t a b o l i c transformations of organic substrates, the reaction free energy is d e t e r m i n e d b y the different b o n d energies of the reactant(s) a n d product(s), w h i c h i n t u r n are set b y the covalent structure. S u c h transformations cannot be m o d i f i e d w i t h o u t c h a n g i n g the c h e m i c a l i d e n t i t y of the reactant, a change that w o u l d r e q u i r e n e w m e t a b o l i c pathways. F o r redox p r o t e i n s , h o w e v e r , the redox potentials that set the m e t a b o l i c free energy are c o n t r o l l e d b y subtle conformational effects i n the s u r r o u n d i n g p r o t e i n , so that the reaction free e n e r g y m a y b e a l t e r e d w i t h o u t r e q u i r i n g the creation of any n e w m e t a b o l i c pathways. O n e final caveat is necessary. To make m e a n i n g f u l measurements o f i n v i v o activity, the strains c o m p a r e d m u s t b e genetically i d e n t i c a l i n a l l r e spects. T h e exception w o u l d be the c y t o c h r o m e c gene itself, w h i c h s h o u l d b e present as a single-copy gene i n t e g r a t e d into the correct p o s i t i o n o n the yeast c h r o m o s o m e . T h e s e r e q u i r e m e n t s have not b e e n m e t i n several at t e m p t e d studies of i n v i v o f u n c t i o n . U n f o r t u n a t e l y , i t is i m p o s s i b l e to o b t a i n any quantitative e s t i m a t i o n of i n v i v o f u n c t i o n (the " d i f f e r e n t i a l specific ac t i v i t y " ) i n these constructs w i t h m u l t i c o p y genes, l i k e r e p l i c a t i n g p l a s m i d s .

Factors That Affect the Redox Potential of Cytochrome c A l t h o u g h c y t o c h r o m e c serves as a p a r a d i g m for m a n y s t r u c t u r e - f u n c t i o n relationships i n proteins (9), the factors c o n t r o l l i n g the basic redox p r o p e r t i e s In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, J., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1991.

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of c y t o c h r o m e c r e m a i n elusive (10, 11). Factors that have b e e n suggested as i m p o r t a n t i n c l u d e h e m e " e x p o s u r e " (i.e., local d i e l e c t r i c e n v i r o n m e n t ) (12-14), axial l i g a t i o n (15), surface-charge d i s t r i b u t i o n (16), a n d redox statespecific h y d r o g e n - b o n d i n g patterns (J 7,18) specifically i n v o l v i n g the H - b o n d n e t w o r k b e t w e e n a h e m e propionate, A s n 57, A r g 38, a n d a n i n t e r s t i t i a l H 0 m o l e c u l e . T h e s e factors are carefully c o n t r o l l e d ; i n a l l species, r a n g i n g 2


from yeast to plants to h u m a n s , the redox p o t e n t i a l of c y t o c h r o m e essentially i n v a r i a n t at E° = 2 6 5 ( ± 1 0 ) m V .

c is

W i t h the advent of p r o t e i n - e n g i n e e r i n g techniques (12-14, 17-21), i t has b e c o m e possible to investigate the roles p l a y e d b y i n d i v i d u a l a m i n o acids i n c o n t r o l l i n g the redox p o t e n t i a l a n d redox activity of this p r o t e i n .

Results and Discussion Charge Mutants. Structural Studies. W e first c o n s i d e r the effects of single-charged residues. I n early w o r k , Rees (22) suggested that the c o u l o m b i c p o t e n t i a l b e t w e e n a surface-charged residue a n d the h e m e c o u l d m o d u l a t e the redox p o t e n t i a l a n d p r o v i d e d some data o n c h e m i c a l l y m o d i f i e d p r o t e i n s i n support of this suggestion. T h i s analysis was q u e s t i o n e d b y M o o r e a n d co-workers (JO, 11), w h o p o i n t e d out several possible pitfalls i n the analysis b u t d i d not d i r e c t l y address the data of Rees (22). W e have isolated a n d extensively c h a r a c t e r i z e d (19-21) single-site replacements i n yeast isoi - c y t o c h r o m e c for several of the e v o l u t i o n a r i l y invariant lysines that s u r r o u n d the h e m e a n d are thought to b e functionally significant. T h e derivatives investigated h e r e i n c l u d e L y s 32 —» L e u , H e , A r g 18 —» H e , a n d L y s 77 —» A r g , A s p . E a c h of these replacements support aerobic r e s p i r a t i o n i n v i v o a n d have b e e n extensively characterized i n v i t r o . T h e reactivities of these derivatives w i t h various p h y s i o l o g i c a l partners of c y t o c h r o m e c have b e e n r e p o r t e d p r e v i o u s l y : T h e M i c h a e l i s parameters, K (related to b i n d i n g ) , a n d the m a x i m u m rate, K , v a r y less t h a n t w o f o l d for a l l these derivatives relative to w i l d t y p e . S t r u c t u r a l characterizations i n solution have b e e n c a r r i e d out b y U V - v i s i b l e spectroscopy, c i r c u l a r d i c h r o i s m , a n d N M R spectroscopy. N o significant changes i n structure c a n b e o b s e r v e d b y any of these t e c h n i q u e s . W i t h i n e x p e r i m e n t a l e r r o r , a b s o r p t i o n wavelengths a n d relative e x t i n c t i o n coefficients (e.g., Soret, 695) for each of these derivatives are the same as w i l d t y p e i n b o t h the v i s i b l e spectra a n d the c i r c u l a r d i c h r o i c ( C D ) s p e c t r u m . T h e positions of the v e r y s t r u c t u r e sensitive h y p e r f i n e shifted h e m e resonances are also u n p e r t u r b e d b e t w e e n w i l d t y p e a n d these charge mutants. m

c a t

Redox Potential. T h e potentials of these a n d other mutants have b e e n d e t e r m i n e d b y p o t e n t i o m e t r i c titration w i t h [Co(II)(terpyridine) ]Cl2", o p tically transparent t h i n - l a y e r e l e c t r o c h e m i s t r y , a n d d i r e c t e l e c t r o c h e m i s t r y o n p a r t i a l l y o x i d i z e d graphite electrodes. T h e redox potentials so o b t a i n e d 2


11.


183



for single (Lys - » neutral) or d o u b l e (Lys —» Asp) charge changes at i n d i v i d u a l residues are l i s t e d i n Table I. O n l y v e r y s m a l l changes i n p o t e n t i a l , w h i c h can l e a d to e i t h e r a decrease or an increase i n E°, are o b s e r v e d . T h e s e results support the analysis b y M o o r e a n d co-workers (JO, 11) of charge effects. I n d i r e c t l y , the data i m p l y that any charge effects are s c r e e n e d b y a h i g h effective d i e l e c t r i c m e d i u m b e t w e e n the l y s i n e N e a n d h e m e i r o n .

Table I. Cyclic Voltammetric Results of the C y t c Mutants at Modified G o l d and Edge-Plane Graphite Electrodes Cyt c Mutant Cys 102 Thr (70 μΜ) Asn 52 I l e - C y s 102 Thr (99 μΜ) Asn 52 A l a - C y s 102 Thr (80 μΜ) Lys 72 Asp (143 μΜ) Lys 27 G i n (110 μΜ) Arg 13 Ile (181 μΜ)

WM)0

AE

d

e

d

e

d

e

d,e

0.31

(±5mV)

b

D

—

d,e

0.49

—

68

d,e

(±5mV)

+ 33

— e

m

—

74

e

E

+ 41 + 39 -10 -10 + 18 + 12 + 31

70 80 67 68 70 152 66

0.22 0.53 0.36 0.73 0.31 QA7 0.44 e

+ 62

—

—

Results for cyt c are in 100 mM KC1/10 mM HEPES, pH 7.4; at modified gold, 0.125 cm ; and edge-plane graphite (EPG) electrodes, 0.148 cm . "Anodic peak current measured at 20 mV s" . Peak separation measured at 100 mV s" . "Associated with modified gold electrodes. Associated with E P G electrodes. Deterioration of the voltammograms prevented precise measurements. NOTE:

2

2

1

6

1

rf e

Η - B o n d i n g M u t a n t s : A s n 57 - » l i e , A l a .

T h e second class of m u

tants e x a m i n e d are i n v o l v e d i n a c o m p l e x h y d r o g e n - b o n d

n e t w o r k that i n

cludes the h e m e propionate a n d a n i n t r i n s i c water m o l e c u l e ( F i g u r e 2). S e v e r a l residues participate i n this n e t w o r k , i n c l u d i n g A r g 38, A s n 52, T y r 67, a n d T h r 78. R e c e n t l y , C u t l e r et a l . (17) r e p o r t e d that m u t a t i o n of A r g 38 can result i n significant shifts i n E° (up to 50 m V ) i n the d i r e c t i o n p r e d i c t e d b y an electrostatic m o d e l d e v e l o p e d b y C u t l e r et a l . (17). S u b sequently, L u n t z et a l . (23) demonstrated that the r e p l a c e m e n t of T y r 67 b y P h e leads to a 4 0 - m V decrease i n E°. W e have f o u n d that replacements at position 52 l e a d to s i m i l a r changes. Structural Studies. S o l u t i o n structural characterization of the A s n 52 —» (He, Ala) mutants shows that there are no significant changes ( H e mutant u s i n g X - r a y diffraction t e c h n i q u e s ,



184


Figure 2. Hydrogen-bond

network in left, native (N 52) and right, the lowpotential mutant (I 52).

w h i c h show that t h e r e are no significant changes i n the m a i n - c h a i n positions b e t w e e n the m u t a n t a n d w i l d - t y p e proteins (24). H o w e v e r , one i m p o r t a n t change that has o c c u r r e d is the d i s p l a c e m e n t of an i n t e r n a l H 0 b y the s u b s t i t u t e d H e side c h a i n . W a t e r d i s p l a c e m e n t has also l e d to significant alterations of the h y d r o g e n - b o n d i n g p a t t e r n i n this r e g i o n ( F i g u r e 2). C l e a r l y the h e m e (electronic) e n v i r o n m e n t is a l t e r e d b y the A s n 57 —» H e , A l a r e p l a c e m e n t s , as i n d i c a t e d b y these structural analyses a n d significant shifts i n the positions of the h e m e h y p e r f i n e shifted resonances, w h i c h are ex c e e d i n g l y sensitive to small p e r t u r b a t i o n s i n the e l e c t r o n - d e n s i t y d i s t r i b u t i o n o f the h e m e . 2

Redox Potentials. T h e A s n —> He r e p l a c e m e n t leads to a significant 5 0 m V shift i n E°, f r o m 270 to 2 2 0 ( ± 5 ) m V . A similar shift was r e p o r t e d b y L u n t z et a l . (23) for the r e p l a c e m e n t of T y r 67 —» P h e . T h e y u s e d this shift to p r e d i c t that loss o f any o f the critical Η-bonding t r i a d (Tyr 67, A s n 52, or T h r 78) c o u l d l e a d to loss of the i n t e r n a l w a t e r m o l e c u l e , w i t h consequent effects o n p r o t e i n stability ( w h i c h increases w h e n the i n t e r n a l H 0 is r e placed) a n d redox p o t e n t i a l ( w h i c h decreases). T h e results f r o m the H e 52 m u t a n t , i n w h i c h the i n t e r n a l w a t e r is k n o w n to be absent, support this p r e d i c t i o n . H o w e v e r , k e y structural studies b y H i c k e y et a l . (24) of the T y r 67 —> P h e m u t a n t do not show w a t e r d i s p l a c e m e n t ; i n fact, an extra w a t e r is a d d e d to the cavity to replace the lost T y r O H . A s i m i l a r shift is f o u n d for the A l a 57 m u t a n t , for w h i c h E° = 2 3 0 ( ± 1 0 ) m V . T h u s , regardless o f the steric b u l k o f the r e p l a c e m e n t , a s i m i l a r redox p o t e n t i a l is o b s e r v e d . T h i s s i m i l a r i t y suggests that the i n t e r n a l w a t e r is i n d e e d a k e y d e t e r m i n a n t of c y t o c h r o m e c redox potentials. 2



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In Vivo Effects of Redox Potential on Growth. W i t h the c o m b i n e d structural a n d redox data i n h a n d , the H e 52 mutants w e r e chosen as t h e best candidates for testing the effect of redox p o t e n t i a l o n redox m e t a b o l i c flux (and, t h e r e b y , c e l l v i a b i l i t y a n d growth). G i v e n the e v o l u t i o n a r y i n v a r iance of redox p o t e n t i a l over a l l eukaryotes, it is logical to anticipate that a 4 0 - m V decrease i n c y t o c h r o m e c p o t e n t i a l m i g h t adversely affect e l e c t r o n transport rate, a l t h o u g h the m a g n i t u d e of such an effect is difficult to a n t i c ipate. F o r any effect to b e o b s e r v e d , m e t a b o l i c conditions are r e q u i r e d for w h i c h c y t o c h r o m e c is i n v o l v e d i n one or m o r e steps that are rate-determ i n i n g i n o v e r a l l m e t a b o l i s m . S u c h conditions are u n u s u a l . H o w e v e r , d e t a i l e d e x p e r i m e n t s o n the genetic regulation of c y t o c h r o m e c have s h o w n that w h e n yeast is g r o w n o n a synthetic m e d i u m that contains lactate as the sole n o n f e r m e n t a b l e c a r b o n source, t h e n net m e t a b o l i s m a n d g r o w t h d i r e c t l y d e p e n d o n c y t o c h r o m e c (25). B y c o n t r o l l i n g the gene sequence u p s t r e a m of c y t o c h r o m e c, it is possible to regulate the t r a n s c r i p t i o n of the c y t o c h r o m e c gene a n d t h e r e b y to regulate the a m o u n t of c y t o c h r o m e c p r o d u c e d i n v i v o . T h e a m o u n t of c y t o c h r o m e c p r o d u c e d can b e q u a n t i t a t i v e l y assayed i n intact yeast cells b y m i c r o s p e c t r o p h o t o m e t r i c measurements of the 5 0 0 n m absorbance d u e to r e d u c e d c y t o c h r o m e c. E x p e r i m e n t s have s h o w n that w h e n the total c e l l u l a r l e v e l of c y t o c h r o m e c is m o d i f i e d b y t r a n s c r i p t i o n a l c o n t r o l , the g r o w t h o n lactate is coregulated i n a monotonie fashion. F i g u r e 3 shows such results. T h e s e results suggest that, u n d e r specific m e t a b o l i c conditions, c y t o c h r o m e c is i n v o l v e d i n a m e t a b o l i c step that is partially rated e t e r m i n i n g for o v e r a l l g r o w t h . W i t h these k e y controls i n h a n d , i t r e m a i n e d o n l y to compare the g r o w t h o n lactate of essentially i d e n t i c a l yeast strains, w h i c h differ o n l y i n the sequence of c y t o c h r o m e c : w i l d type ( N 52) a n d m u t a n t (I 52). T h e i n i t i a l results are q u i t e s u r p r i s i n g ( F i g u r e 4). T h e e v o l u t i o n a r y i n variance of the c y t o c h r o m e c redox p o t e n t i a l has b e e n u s e d to argue that the value of 270 m V critically regulates c e l l u l a r e l e c t r o n transport. T h e 5 0 m V shift of the H e 52 m u t a n t s h o u l d l e a d to a significant change i n the effective p o p u l a t i o n of (oxidized/reduced) c y t o c h r o m e c a n d thus m i g h t d i m i n i s h the g r o w t h rate. T h i s change is not observed. T h e strain that contains the I 52 m u t a n t does not g r o w p o o r l y ; it actually appears to g r o w at a rate that equals or s l i g h t l y exceeds that of the w i l d - t y p e strain! A p p a r e n t l y , the standard redox potentials d e t e r m i n e d i n v i t r o b y c o n v e n t i o n a l e l e c t r o c h e m i c a l methods do not c o n t r o l the rates of e l e c t r o n flow i n v i v o u n d e r m e t a b o l i c conditions i n w h i c h c y t o c h r o m e c is i n v o l v e d i n a r a t e - d e t e r m i n i n g m e t a b o l i c step. F u r t h e r m o r e , extensive e x p e r i m e n t s b y V a n d e r k o o i (26), M a g n e r (27), C h a n c e a n d W i l l i a m s (28), a n d others strongly indicate that this s u r p r i s i n g result is not d u e to a s i m p l e m o d u l a t i o n of the c y t o c h r o m e redox p o t e n t i a l u n d e r c e l l u l a r conditions. I n d e e d , i n a classic p a p e r , C h a n c e a n d W i l l i a m s (28) s h o w e d that the i n v i t r o p o t e n t i a l of c y t o c h r o m e c closely agrees w i t h the p o t e n t i a l m e a s u r e d for c y t o c h r o m e c i n



186


8

time hours Figure 3. Growth curves on lactate minimal media measured by Klett techniques for Saccharomyces yeast containing different intracellular cytochrome c concentrations (controlled genetically). The monotonie dependence of growth on cytochrome c suggests "autogenous regulation". In chemical terms, cytochrome c is involved in one of the rate-determining steps in overall metabolism. intact m i t o c h o n d r i a . W e are also rather certain that this result is not u n i q u e to the H e 52 variant, o n the basis of l i m i t e d qualitative observations i n o u r laboratory a n d others for other c y t o c h r o m e c mutants of altered redox p o tentials (e.g., A l a 52, P h e 67, a n d A l a 38).

Discussion Is there any s i m p l e c h e m i c a l explanation for the c o u n t e r i n t u i t i v e result that c h a n g i n g the e v o l u t i o n a r i l y i n v a r i a n t redox p o t e n t i a l of c y t o c h r o m e c b y a single-site m u t a t i o n does not d i m i n i s h its i n v i v o activity? W e suggest two possible explanations that d e p e n d o n s i m p l e c h e m i c a l kinetics arguments. T h e first (and the most obvious) is that, u n d e r these specific a n d u n u s u a l



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Figure 4. Growth rates for wild type (E° = 270 mV) and the He 52 mutant (E° = 220 mV). The mutant reproducibly grows better than the wild type under these conditions. m e t a b o l i c c o n d i t i o n s , the effective redox p o t e n t i a l of the electron-transport system is c o n t r o l l e d " u p s t r e a m " of c y t o c h r o m e c, so that, for e x a m p l e , c y t o c h r o m e c is > 9 9 % r e d u c e d u n d e r steady-state m e t a b o l i c c o n d i t i o n s . I f so, the redox p o t e n t i a l of c y t o c h r o m e c w i l l b e less i m p o r t a n t i n d e t e r m i n i n g the net flux of the electrons. T h e flux w o u l d still b e sensitive to the total a m o u n t of c y t o c h r o m e c, as observed. A second, m o r e specific explanation is possible. F o r yeast to g r o w o n lactate, lactate m u s t b e o x i d i z e d to p y r u v a t e b y u s i n g the yeast lactate dehydrogenase (cytochrome fc ). T h i s e n z y m e uses c y t o c h r o m e c as a specific "cofactor" for oxidation: x

2

2Fe

3 +

+ cyt c cyt &

2(red)

—» cyt fe

2(ox)

4- 2 F e cyt c n

cyt b ( ) + lactate —» cyt fe ( d) + p y r u v a t e 2

ox

2 re

w h e r e r e d is r e d u c e d a n d ox is o x i d i z e d . I n earlier w o r k (29), w e s t u d i e d


188


Figure 5. Dependence on A G of the rate constant for the reaction of (M)cytochrome c [M is Fe , Zn", or (H ) ] with cytochrome b (lactate de hydrogenase). The top line shows the de pendence predicted by Marcus theory for λ = 1 eV (as in Figure 1 ). The essential free energy independence of rate sug gests conformational gating. The rate-de termining step is not electron transfer, but a prior conformational change. m


+

2

2

the d e p e n d e n c e of the electron-transfer rate o n redox p o t e n t i a l w i t h i n the c y t o c h r o m e c - c y t o e h r o m e b c o m p l e x ( F i g u r e 5). O n the basis of M a r c u s t h e o r y a n d results for the analogous c y t o c h r o m e c - c y t o c h r o m e fo c o m p l e x , w e e x p e c t e d to see a strong d e p e n d e n c e of rate o n A G . Instead, w e f o u n d that the electron-transfer rate was i n d e p e n d e n t of reaction free energy. T h e simplest explanation for this i n i t i a l l y s u r p r i s i n g result is that the e l e c t r o n transfer step, p e r se, is not the r a t e - d e t e r m i n i n g step of the reaction. Instead, a slow conformational change i n the p r o t e i n c o m p l e x precedes the fast elec tron-transfer step. S u c h conformationally c o n t r o l l e d reaction rates, d u b b e d "conformational g a t i n g " b y H o f f m a n a n d R a t n e r (30), have b e e n extensively discussed theoretically. C o n s i d e r the p o s s i b i l i t y that the c y t o c h r o m e c - c y t o c h r o m e b reaction is partially r a t e - d e t e r m i n i n g for g r o w t h i n lactate. T h e n the g r o w t h rate can d e p e n d o n l y o n the total c y t o c h r o m e c c o n c e n tration. E q u a l l y c l e a r l y , o n the basis of i n v i t r o studies, a change i n the redox p o t e n t i a l of c y t o c h r o m e c w i l l not d i m i n i s h the rate of this k e y step because the rate is c o n t r o l l e d not b y an electron-transfer b a r r i e r associated w i t h E° b u t b y a conformational b a r r i e r . A t present these suggestions m u s t b e c o n s i d e r e d r a t h e r speculative. H o w e v e r , t h e y do suggest f u r t h e r e x p e r i m e n t s . F o r e x a m p l e , e i t h e r of these explanations p r e d i c t that other m e t a b o l i c pathways (e.g., g r o w t h o n glucose) s h o u l d result i n different g r o w t h - l i m i t i n g steps, c o r r e s p o n d i n g to different m e t a b o l i c pathways w i t h c o r r e s p o n d i n g l y different steady-state redox levels. T h e s e c o m m o n metabolic states p r e s u m a b l y favor the (higher) c y t o c h r o m e c p o t e n t i a l f o u n d i n the w i l d - t y p e p r o t e i n . T h i s p r e d i c t i o n is b e i n g tested. 2

5

0

2

Acknowledgments W e gratefully acknowledge c o n t i n u i n g interactions w i t h A . G . M a u k , M . S m i t h , a n d E . M a r g o l i a s h . T h i s w o r k was s u p p o r t e d b y N a t i o n a l Science F o u n d a t i o n grants to G e o r g e M c L e n d o n a n d F r e d S h e r m a n , the N a t i o n a l Institutes of H e a l t h ( G M 33881 G M ) , a n d a grant to G a r y B r a y e r f r o m the M e d i c a l R e s e a r c h C o u n c i l of C a n a d a .


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Electron Transfer in Inorganic, Organic, and Biological Systems

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