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Metalloproteins and Electron Transfer S. K . C H A P M A N , D . M. DAVIES, A . D . W A T S O N ,
and A. G. S Y K E S
The University, Newcastle-upon-Tyne, Department of Inorganic Chemistry, Newcastle-upon-Tyne NE1 7 R U England
Studies on 1:1 electron-transfer reactions of metalloproteins with inorganic complexes are, in a number of cases, at a stage where the site or sites on the protein at which electron transfer occurs can be specified. Results obtained for the blue Cu protein plastocyanin are considered here in detail as illustrative of different approaches yielding relevant information. The reduction of plastocyanin PCu(II) with cytochrome c(II) is also considered as an example of a protein-protein reaction. It has been possible in the latter to define the reaction sites of the two proteins with respect to each other at the time of electron transfer. M e t a l l o p r o t e i n s f a l l i n t o three main s t r u c t u r e c a t e g o r i e s depending on whether the a c t i v e s i t e c o n s i s t s of a s i n g l e coordinated metal atom, a metal-porphyrin u n i t , or metal atoms i n a c l u s t e r arrangement. In the context of e l e c t r o n - t r a n s f e r m e t a l l o p r o t e i n s , the blue Cu p r o t e i n s , cytochromes, and f e r r e doxins r e s p e c t i v e l y are examples of these d i f f e r e n t s t r u c t u r e types. A t t e n t i o n w i l l be confined here mainly to a d i s c u s s i o n of the r e a c t i v i t y of the blue Cu p r o t e i n p l a s t o c y a n i n . Reactions of cytochrome c are a l s o considered, with b r i e f mention o f the [2Fe-2S] f e r r e d o x i n , and high p o t e n t i a l Fe/S p r o t e i n [HIPIP]. I t i s timely to review the r e a c t i v i t y of p l a s t o c y a n i n i n the l i g h t of recent aqueous s o l u t i o n s t u d i e s , and the elegant s t r u c t u r a l work of Freeman and colleagues on both the PCu(I) and PCu(II) forms (1,2). P l a s t o c y a n i n now ranks alongside c y t o chrome c (3) as the e l e c t r o n - t r a n s f e r m e t a l l o p r o t e i n f o r which there i s most s t r u c t u r a l information. The aim i n s o l u t i o n s t u d i e s on m e t a l l o p r o t e i n i s to be able to say more about i n t e r m o l e c u l a r e l e c t r o n t r a n s f e r processes, f i r s t of a l l by studying outer-sphere r e a c t i o n s with simple inorganic complexes as redox partners. With the information (and experience) gained i t i s then p o s s i b l e to turn to p r o t e i n p r o t e i n r e a c t i o n s , where each reactant has i t s own complexities
0097-6156/83/0211-0177$06.25/0 © 1983 American Chemical Society
INORGANIC CHEMISTRY: TOWARD THE 21ST CENTURY
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178
and r e a c t i o n s are correspondingly more d i f f i c u l t to assess. A f u l l e r understanding of r e a c t i o n s i n v o l v i n g p h y s i o l o g i c a l p r o t e i n partners i s the ultimate o b j e c t i v e . In a d d i t i o n to the a c t i v e s i t e chemistry of each p r o t e i n , the i d e n t i f i c a t i o n of a s i t e (or s i t e s ) on the p r o t e i n surface at which e l e c t r o n t r a n s f e r occurs, the nature and s p e c i f i c i t y of such f u n c t i o n a l s i t e s , the distance over which e l e c t r o n s are t r a n s f e r r e d , and the manner i n which e l e c t r o n s are t r a n s f e r r e d ( i n t e r v e n i n g groups?) are r e l e v a n t . Multimetal p r o t e i n s such as cytochrome oxidase with, more than one a c t i v e s i t e are not considered here. Intramolecular e l e c t r o n t r a n s f e r i s an a d d i t i o n a l feature with such p r o t e i n s . Function of P l a s t o c y a n i n . P l a s t o c y a n i n (M.W.10,500, 99 amino-acid residues) occurs i n a l l higher p l a n t s as w e l l as green and blue-green algae (104 amino acids) (4). I t has a r e l a t i v e l y w e l l defined f u n c t i o n i n photosynthetic e l e c t r o n t r a n s f e r and acts as an oxidant f o r membrane bound cytochrome f (M.W.33,000), and as a reductant f o r P700 which i s the double c h l o r o p h y l l pigment of photosystem 1. The Cu i n p l a s t o c y a n i n has a C u ( I I ) / Cu(I) r e d u c t i o n p o t e n t i a l of 370 mV at pH 7, which i s between that of cytochrome f (360 mV) and P700 (520 mV). From the aminoa c i d composition cytochrome f i s estimated to have a charge of -30 at pH 7 05). Structure of P l a s t o c y a n i n . The s t r u c t u r e of PCu(II) i s now known to 1.6A r e s o l u t i o n , (2). The p r o t e i n contains a s i n g l e Cu atom with a d i s t o r t e d t e t r a h e d r a l c o o r d i n a t i o n geometry. I t i s coordinated to the imidazole N40 residues) of plastocyanins from higher p l a n t s i t appears that s i x t y residues are i n v a r i a n t and 7 are c o n s e r v a t i v e l y s u b s t i t u t e d (2,7). With three a l g a l plastocyanins included there are 39 i n v a r i a n t or c o n s e r v a t i v e l y s u b s t i t u t e d groups. I t i s b e l i e v e d that the same s t r u c t u r a l features apply to the whole f a m i l y , and that h i g h l y conserved residues are an i n d i c a t i o n of f u n c t i o n a l s i t e s on the p r o t e i n surface. The upper hydrophobic and right-hand-side surfaces are b e l i e v e d to be p a r t i c u l a r l y r e l e v a n t i n t h i s context, the l a t t e r i n c l u d i n g four consecutive 2
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CHAPMAN E TAL.
Metalloproteins
and Electron
Transfer
Figure 1. Structure of plastocyanin (2) showing the positions of α-carbon atoms of amino acid residues. The active site and positions of the conserved (plant) negative patch (42-45) and Tyr 83 are indicated (Φ).
Figure 2.
The Cu active site of plastocyanin PCu(II) (2).
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n e g a t i v e l y charged amino a c i d s (which form a negative patch 4 2 45) as w e l l as Tyr-83. P l a s t o c y a n i n from the blue-green a l g a l source Anabaena v a r i a b i l i s has no negative patch (only 42 i s a c i d i c ) Ç 4 ) . The e x t r a amino acids (104 compared to 99) are incorporated i n lower s e c t i o n s of the s t r u c t u r e remote from the active s i t e . Rate Constants and R e a c t i v i t y . E l e c t r o n - t r a n s f e r r e a c t i o n s of p l a s t o c y a n i n (and other m e t a l l o p r o t e i n s ) are so e f f i c i e n t that only a narrow range of redox partners (having small d r i v i n g force) can be employed. Rates are i n v a r i a b l y i n the stoppedflow range, Table I . Unless otherwise stated p a r s l e y p l a s t o c y a n i n
Table I Summary of r e d u c t i o n p o t e n t i a l s and r a t e constants f o r r e a c t i o n s with p l a s t o c y a n i n PCu(I) and PCu(II) at 25C, pH 7-8, I = 0.10 M(NaCl). E«Cytochrome c ( I I I ) / ( I I ) Ru(NH ) py3+,2+ PCu(II)/PCu(I) Co(phen) 3+,2+ Co(bipy) 3+,2+ Co(dipic)2", ~ Fe(CN) 3-,43
a
k . , oxid (mV) (M-1 s-1) 260 260 273 10^ c 4.2 χ 10* 273 370 3.0 χ 103 370 313 370 400 510 410 9.4 χ 104 10* 410
5
3
3
2
6
*As oxidant f o r PCu(I) bAs reductant f o r PCu(II) cAt pH 5.8, I = 0.10M (NaClO^ dValue obtained at low [ C o ( p h e n ) kinetics effective.
i s used i n studies described. +
PCu(I) + C o ( p h e n ) 3 — »
3+ 3
] before
Reactions
red (M-1 s-1) 1.45 χ 1θ6 4.2 χ 105
1.9 χ 10*
limiting
as i n (1)
PCu(II) + Co(phen)3
+
(I) 1
1
are monitored at the PCu(II) peak at 597nm (ε 4500 M" cm" ) with the inorganic redox partner i n >10-fold excess. With cytochrome c (M.W. 12,500, 104 residues) the F e ( I I ) form i s r e a d i l y monitored at 417nm (Ac 4 χ 1θ5 M"l cm"l) and the r e a c t i o n i s best studied with PCu(II) i n l a r g e excess, ( 2 ) . Cyt c ( I I ) + PCu(II) — ^
Cyt c ( I I I ) + PCu(I)
(2)
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ET
Metalloproteins
AL.
and
Electron
181
Transfer
Ionic strengths are g e n e r a l l y adjusted to I = 0.10M (NaCl). A range of b u f f e r s (~10" M) has been employed with s a t i s f a c t o r y agreement i n overlap r e g i o n s . Phosphate i s known to a s s o c i a t e with cytochrome c and was t h e r e f o r e avoided i n a l l such s t u d i e s . Care i s r e q u i r e d when studying r e a c t i o n s which are thermodynami c a l l y unfavourable, since i t i s necessary to have high concen t r a t i o n s of redox partner to ensure that the r e a c t i o n proceeds to completion (>95%). I f t h i s c o n d i t i o n i s not met i n c o r r e c t i n t e r p r e t a t i o n can r e s u l t unless a more r i g o r o u s k i n e t i c t r e a t ment i s employed (8). Simple f i r s t - o r d e r k i n e t i c s ( k b ) apply i n very many cases, (3),
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2
Q
Rate
=
k [P]
[C]
s
(3)
i . e . k b = k [ C ] , where [P] and [C] are p r o t e i n and complex con c e n t r a t i o n s r e s p e c t i v e l y . T y p i c a l r a t e constants, k, are l i s t e d i n Table I . Attempts to apply Marcus c o r r e l a t i o n s give a wide range of r a t e constants (50 to 3 χ 106 M"l s ~ l ) f o r the PCu(I) + PCu(II) self-exchange (9). 0
s
L i m i t i n g K i n e t i c s . In some instances f i r s t - o r d e r r a t e constants ( k s ) give a l e s s than f i r s t - o r d e r dependence on the reactant i n l a r g e excess, and (3) no longer a p p l i e s . Instead, i n f o r example the Co(phen>33+ o x i d a t i o n of PCu(I), (4) holds, 0 D
k , obs
=
K k
et
1 + K with Κ and k Ρ + P,
c
c
e t
[C]
(4)
[C]
as defined i n (5) and (6), P, C
(5)
products
(6)
Such behaviour i s i d e n t i f i e d by curvature when k f c i s graphed against [C], F i g u r e 3, (10). Both Κ and k can be obtained from l i n e a r p l o t s of ( k f c ) - l against [C]""l. I f Κ i s small so that K [ C ] « 1 then the k i n e t i c s conform to (3). Under such circumstances i t i s s t i l l appropriate to think i n terms of a two-step process with k = Κ k f I d e n t i f i c a t i o n of (5) - (6) with the i m p l i c a t i o n of long d u r a t i o n or ' s t i c k y c o l l i s i o n s i s important i n that i t could present a means by which low probab i l i t y (long distance?) e l e c t r o n t r a n s f e r could occur. For r e a c t i o n s i n v o l v i n g l a r g e protein-molecules e l e c t r o n t r a n s f e r over long d i s t a n c e s may be a n e c e s s i t y . A number of examples of l i m i t i n g k i n e t i c s have been reported f o r r e a c t i o n s of [2Fe-2S] and 2[4Fe-4S] f e r r e d o x i n s with i n o r ganic complexes (11). Recent stopped-flow work has not however confirmed l i m i t i n g k i n e t i c s f o r the r e a c t i o n of a z u r i n , ACu(I) + 0
O D S
0
s
e
1
s
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INORGANIC C H E M I S T R Y : TOWARD T H E 21ST C E N T U R Y
3
Figure 3. Dependence of first-order rate constants k (25 °C) vs. [Co(phen) *] for the oxidation of plastocyanin PCu(I). Conditions: pH, 7.5 (phos); and I, 0.10 M (NaCl). Key: M, spinach; and A, parsley. (Reproduced from Ref. 10. Copyright 1978, American Chemical Society.) obe
s
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and
Electron
183
Transfer
4
Fe(CN) 3~, and f o r the r e a c t i o n Fe(CN) "" + PCu(II) at pH 7, and a requirement f o r (4) would now seem to be that the o v e r a l l charges on the two reactants are of opposite s i g n . Clearly k = Κ k t a p p l i e s since ΔΗΪ f o r k i n the ACu(I) + Fe(CN) 3r e a c t i o n i s negative implying a composite term with ΔΗ f o r Κ having a negative value. From the amino-acid composition charges on ACu(I) and PCu(II) are estimated to be -1 and -7 r e s p e c t i v e l y at pH 7. The i o n i c strength at which a k i n e t i c study i s c a r r i e d out can of course have an i n f l u e n c e on behaviour and i n t e r p r e t a t i o n . Since each reactant has a s s o c i a t e d with i t an i o n i c atmosphere of opposite charge, Κ w i l l become smaller as the i o n i c strength i n c r e a s e s . L i m i t i n g k i n e t i c s (4), i s l e s s l i k e l y therefore at I = 0.50 M, (12), and conversely more l i k e l y to be detectable at I < 0.10 M. To t e s t f o r l i m i t i n g k i n e t i c s r e l a t i v e l y high concentrations of C (
