Electron transfer in proteins

Electron

transfer

Ramy S. Farid, Christopher University

in proteins

C. Moser and P. Leslie Dutton

of Pennsylvania,

Philadelphia,

USA

The past year has seen significant advances in our understanding of electron transfer (ET) events in biological and synthetic protein systems. Experiment and theory have begun to merge towards a more complete picture of the role that protein plays in mediating ET reactions. This review highlights these developments and discusses the roles played by distance, protein medium, driving force, and reorganization energy in controlling ET rates in natural and artificial systems. These parameters, drawn from ET theory, are discussed in terms of their natural selection to govern biological ET. We find that distance, driving force, and reorganization energy are the main determinants of ET rates and directional specificity. Another parameter that can have significant effects on rates over physiological distances is the structure of the intervening medium between the electron donor and acceptor cofactors. There is nevertheless no clear evidence that the effect of distance on ET is modified by the intervening protein structure so as to favor productive forward ETs over energy-wasting reverse reactions in key biological ET systems. Current

Opinion

in Structural

Biology

tors were treated classically (i.e. ignoring the possibility of nuclear tunneling) by Marcus over 30 years ago. Marcus predicted a striking dependence of ET rates on the reaction free energy, - AG” [l] :

Introduction

Electron transfer (ET) proteins comprise a class of proteins that are vital components of respiratory and photosynthetic chains and many enzymes. Common to these proteins are cofactors that in their ground or excited state donate or accept electrons. ET>may occur between cofactors positioned at distances that range from virtual contact to as much as 2OA within a single protein or across the boundaries of proteins that form transient complexes. A detailed description of the role that the protein medium plays in mediating ET is required before we,can understand the function and natural evolution of biological ET systems.

Electron-transfer

ket

ket = (+2Xk,Z’)‘~

For long-distance ET between weakly coupled donor and acceptor, the non-adiabatic description of Fermi describes the relationship between the ET rate and electronic and nuclear terms, thus:

V, is the electronic coupling between donor and acceptor and contains information concerning the nature of the medium and the donor-acceptor separation. The nuclear-geometry-dependent Frank Condon (F.C.) fac-

@

Current

V,12(l?C.)

[VR]2exp -(AGO -X)2,4XkgT

1

(2)

As isolated orbital wavefunctions decay exponentially with distance, kt is also expected to fall off approximately exponentially with some measure of the distance (R) between donor and acceptor (usually, but not con-

Abbreviations cyt-cytochrome; Biology

= $1

Inspection of the classical Marcus expression (Eqn 2) shows that the ET rate (k& increases when - AG” is less than the reorganization energy, h, reaches a maximum value when - AG” = h, and then actually decreases when - AG’ > h. The latter surprising prediction, the so-called ‘inverted region’, has only recently received experimental verification (e.g. [ 2,3] >, but its generality remains uncertain. In addition to the classical treatment of the nuclear terms, several theories [4,5] have emerged that take into account the quantum nature of the reactant and product states and introduce the quantum energy, ho to the harmonic oscillator potential surfaces of Marcus. These theories essentially modify the shape of driving force ( - AG”) versus log(rate) plots, tending to moderate the steepness of the inverted region.

theory

bpy-bipyridine;

1993, 3:225-233

Ltd

W-electron ISSN

0959-440X

transfer. 225

226

Theory sistently,

and

simulation

taken as the shortest edge-to-edge distance, &‘I: kef = k,e.rp[+(R

-&)I

(3)

(a) log b,,,x p-9

The rate of exponential decay with K is given by a COefficient, p, which describes the overall el?ect of the intervening medium in propagating the wavefunctions (Eqn 3). The contribution of the medium to the value of p and the implications of this value in the control of biological ET events has been a topic of much discussion in the literature’during the past year.

Protein medium: pathways?

organic

glassy

solvent

or tunneling

We recently proposed a hypothesis that nature uses only distance, modulated by driving force and reorgnnization energy, to control the rates of ET [6*-l. This was substantiated by the observation that a plot of the logarithm of free-energyoptimized ET rates [ IogCk,,,,,)] versus distance yielded a single line for all biological ;111d some semibiological systems (Fig. la). The line that a~commodates these disparate reactions is described by an average p of 1.4A- 1 and an intercept at \m der Wnals contact of - 1013 s- 1, A similar analysis was made for almost 30 covalently linked donor-acceptor model compounds. These data IGelded a line with an average p of O.?k * (Fig. 1b), demonstrating that the coupling between donor and acceptor in directly linked systems is enhanced relative to proteins. The typical mix of covalent links and interstitial space between redox centers in proteins was seen as the origin of a value of p inter. mediate between that of directly linked redox centers (0.7 A- 1) and that anticipated for vac~~u~~~ (2.8 p\- 1 ). A similar intermediate value for p ( 1.2A- 1) was found from ET investigations in organic glassy solvent [ 71, The implication in this work is that the p found in proteins, although contributing to the rates observed, is a characteristic that has not been deliberately manipulated in nature. A similar generic role was suggested for the nuclear term 50. In this hypothesis, the protein is seen to pla) a vital role in providing a structural framework for the cofactors, and the chemical and physical environments that establish the AG” and h values. But, as far as p and ~IW are concerned, the hypothesis suggests that the protein presents a medium for ET with properties similar to those found in organic glasses. These observatidns allowed us to develop an empirical approsiriIat.ion for intraprotein ET transfer (Eqn 4) and for ET in covalently bridged model complexes (Eqn 5) which has the simple Gaussian form of a Marcus-like expression and includes a log relationship between the rate and donor-acceptor distance using the apparently generic values of b and-iiw for the respective systems:

logbl = 15.2 - 0.61R - 3.1(AG - X)*/x

(4)

logkt = 13.3 - 0.36R - 3.1(AG - X)*/x

(5)

(b) log &,,a, b-‘)

”

I

I

I

5

10

15 Distance

$1

Fig. 1. (a) Graph of free-energy-optimized ET rate (k,,,) versus donor-acceptor distance for ET reactions in the photosynthetic reaction center (0 0) and semisynthetic Ru-cytochrome c and Ru-myoglobin to,+). For reactions with limited free energy dependence, error bars span the range of optimal rates calculated from a reasonable range of reorganization energies. The line represents the best fit to the data: p = 1.4 A-1; y-Intercept at van der Waals contact (3.5& = 10’35-1. (b) Graph of free-energyoptimized ET rate versus distance for ET reactions in covalently bridged donor-acceptor complexes featuring either bridges incorporating aromatic groups f A, A 1 or non-aromatic bridges (0,m). Filled symbols are for reactions with extensive free energy dependence, whereas open symbols are for reactions with smaller experimental free energy dependence and therefore more uncertain optimal rates.

An issue important to this model and its verification is the definition of distance: in particular, the identification of :I proper boundary between the donor and acceptor molecules 2nd the molecular groups of the intervening medium. For pragmatic reasons, Moser rf al. [6**] chose the simplest definition of distance. This w;1s the shortest straight-line distance between the centers of atoms at the edges of the donors and acceptors (referred to as Q). The edges of the donors and acceptors were identified only by the atoms that are in conjugation p was and directly involved with the reactions. Thus,
Electron

tion, i.e. as part of the medium. In the case of quinones, only the ring(s) and carbonyls were used to define the redox molecules. In the case of ruthenium complexes attached to proteins via histidine, ail ligands (including the imidazole ring) were considered to be part of the redox cofactor. During the course of this review, we will question the validity of this definition of distance and the impact that it has on current investigations. In a theoretical approach addressing the above issues, Beratan, Bet& and Onuchic [8**,9*] have explored the atomic structure of protein medium between donor and acceptor as a possible means of varying p, and hence modulating the extent to which distance controls ET rates. Calculations revealed that the secondav and tertiary structures of proteins could, in principle, modulate ET rates by up to four orders of magnitude at any fixed distance typical for intraprotein ET reactions. This view highlights the potential of the protein medium to play a significant role in promoting or hindering the path of a transferring electron and hence, if engineered into the structure of the protein, governing the direction of ET in natural systems. The model goes on to identify dominant bonding and non-bonding interactions that might be responsible for the donor-acceptor coupling. Segments of the ‘pathway’ between donor and acceptor are thus characterized as a covalent (C), hydrogen-bonded (H) or through-space (S) interaction. The coupling arising from a single pathway between donor and acceptor is represented as:

where EC= 0.6, ES= O.Gexp[ .- 1.7(R - 1.-i)], and EH = 0.36exp[ - 1.7(R - 2.8)]. A factor of 1.~2 is sometimes introduced into the pre-exponential of the expression for ES to approximate the orientation factors associated with non-bonding interactions. In practice, the mkmum value of vR is chosen to represent the overall V,. An indication of the variance in electronic coupling displayed by tunneling pathways composed of varying proportions of covalent, hydrogen-bonded and throughspace (vacuum) interactions is given in Fig. 2, which represents the result of a pathway search from the heme redox cofactor to all other non-hydrogen atomic sites in cytochiome (cyt) c. The figure correlates interatomic distances with electronic coupIing (masimuni vK) for each hypothetical donor-acceptor pair [9-l. The scatter of two orders of magnitude in rate (and a factor of 10 in electronic coupling) on either side of the best-tit line indicates the potential control that intraprotein interactions have on ET rates. Despite the increased insight into possible efl‘ects on ET rates from intervening protein structure, this theoretical treatment is subject to un certainties regarding donor-acceptor boundaries similar to those described in the hypothesis of Moser et a/, [ 6**], Thus, the two approaches of Beratan, Betts and Onuchic, and Dutton and coworkers acknowledge the existence of substantial local variation in p values. The principle

transfer

Farid, Moser and Dutton

in proteins

-’

I%

(pathway coupling)

*

-3

8

6 4

-5

0

* .

2 ’

I 5

I

I

I

-,I

10

15

20

25

Distance

-7

f/$

Fig. 2. Graph of calculated coupling (and ET rates Ikl) versus distance for pathways between the heme and all non-hydrogen atoms in cyt c. The maximum coupling (0.6) is arbitrarily set at a rate of 4 X 1013s-l. The two orders of magnitude scatter in the cotipling at fixed distance is associated with the predicted four orders of magnitude modulation of ET rate at a fixed donor-acceptor edge-to-edge distance. The solid line represents the best fit to the entire set of couplings for the protein (average !3 = 1.2 A-9, and the dashed line represents the best-fit line to data from Fig. l(a).

points of debate are: can ET rates be modulated fo; a given distance by 0 variances in protein structure, as predicted by the results of Fig. 2; and, in the event that suc11 variations in rates are demonstrated, even for nonphysiological ET reactions, are there any cases in which it is clear that local variations in p have been selected as a natural design feature of functioning biological redos proteins? The first question can be addressed experimentally by attaching donors and acceptors to proteins in positions deliberately chosen to express the predictions of Fig. 2. Answering the second question requires a continued search of ET reactions for natural systems that exhibit physiologically significant ‘deviations’ from the best-fit line of Fig. l(a).

lntraprotein

electron

transfer

Several new studies have been-aimed at addressing the first question above, i.e. can ET rate be modulated by p variances in protein structure? These investigations have utilized natural ET proteins that have, in some cases, been modified by covalently attaching artificial redox complexes to the exterior of the protein; in other cases, artificial reactions have been initiated within the protein between existing redox centers. An investigation has been undertaken in a series of modified cyts c [ 10°*,ll*] in which ET was measured from the reduced heme (Fe’+) to oxidized ruthenium bipyridine (bpy) complexes attached to surface histidines at various positions (Eqn 8):

(7) Ru(bpy)2(im)(HisX)3+

-Fe*+

227

228

Theory

and simulation

The Ru3+ -F$+ -cyt c species was generated by the reaction in Eqn 7. I

of distance yields k,, values that fall below the best-fit line of Fig. l(a). Most notable is the His72-modified cyt c with a reported edge-to-edge distance of 8.4A (measured From the edge of the imidazole ligand to the S atom of Met80), and which exhibits a k,, four orders of magnitude smaller than the line of Fig. l(a). Thus, it appears that the protein medium between His72 and the heme is associated with a much larger p value than the average value for the entire protein.

The driving force (-AGO = 0.74 eV> for these four reactions is presumed to be cdnstant and nearly equal to the reorganization energy (h = 0.8 f 0.2 eV) [ 121, and so the observed kt approximates k,, (see Eqn 2). (Although the uncertainty in h appears to be large, the actual value has only a small etfect on the extrapolated values of k,,,, because the driving forces are relatively large.) Table 1 summarizes the ET parameters for the four proteins studied. The donor-acceptor edge-to-edge distances in this work [lo**] were measured from modeled structures based on the X-ray crystal structure of cyt c from the Ru complex (including the protein imidazole l&and) to the closest atom of the heme group or atoms of the axial ligands, i.e. the imidazole ring of His18 or the S atom of Met80). Table 2 shows that this deiinition

The Beratan and Onuchic model has been applied to this system and appears to account for these deviations in local p values. In His72-modified cyt c, the pathway analysis yields a connectivity that involves eight covalent bonds and a through-space gap of 3.88% yielding the equivalence of 17.6 o-bond units (the coupling across 3.88A through-space jump equals that of 9.6 covalent bonds) or a ‘o-tunneling length’ (01) of 24.6A (17.6 x 1.4A). This pathway does not include the bond between the Met80 S atom and the heme Fe atom, for which p is considered to be zero. Figure 3 shows a plot of loI3 4llax versus this al for His72 and the other reactions; the line (slope = 0.71A-1) correlates well with the data, including a one-bond limit set at 3 x 1012 s-1. This analysis supports the idea that local l3 variations can have a significant influence on intraprotein ET rates.

Ru(bpy)z(im)(HisX)3+-Fe

2+

ket -

Ru(bpy)&r~)(HisX)~+ -Fe3+ x = 33, 39,62,72

Table HisX His39 His33 His72 His62

1. ET parameters

for Rufbpy)zfimXHisX)cyt

(4) X IO6 (3) X 106 (3) x 105 (2) x 104

‘Rate of Ef from Fe3+ to with an error of fO.2eV. of the axial ligands to the the number of equivalent

Table

2. Comparisons

c.

-AC’ teV1

k,ra (s-1) 3.2 2.6 9.0 1.0

(8)

0.74 0.74 0.74 0.74

hb 0.8 0.8 0.8 0.8

k max (s-1) 3.3 2.7 9.4 1.0

x X x x

106 106 105 104

V,

(cm-?

RC

0.11 0.097 0.057 0.0060

d’ (A)

(A)

12.3 11.1 a.4 14.8

19.6 19.5 24.6 28.8

Rufbpy)zfimlfHisX)s +. (Numbers in parentheses represent the experimental uncertainties.) bAssociated CEdge-to-edge distances were measured from the heme group including 5 atom and imidazole ring protein imidazole ligand of the Ru complex. da-Tunneling lengths were calculated by multiplying o-bonds in the pathway by 1.4 A (see text). Data reproduced from I10~~,11~1.

among

HisX

Predicted log fk,,,,) from Ra

His39 His33 His72 His62

7.7 8.5 10.1 6.2

experimental

ET rates

and

rates

predicted

by the

ReC range (average)

A” (s-1) -1.2 - 2.1 - 4.1 - 2.2

for Rufbpy)z(im)(HisX)-cytc.

Predicted

(A) 12.5-13.2 12.4-13.3 11.6-13.0 14.4-15.5

line of Fig. l(a)

(13.0) (12.8) (12.6) (15.2)

aR taken from Table 1. “Deviations in rate from the line of Fig. l(a) in log units. ’ the heme macrocycle to the protein imidazole ligand of the Ru complex using distances were obtained by measuring R, for all energy allowed conformations cyts c.

log fk,,) from R,

Ab (A)

7.3 7.4 7.5 6.0

-0.8 -1.0 -1.6 - 2.0

CEdge-to-edge distances were measured from the rules established in [6**1. The range of of the surface histidine in modeled tuna heart

Electron

10

15

20

25

30

a-Tunneling length

(A)

Fig. 3. Graph of free-energy-optimized ET rates (k,,,) versus otunneling length (01) for Rufbpy)zfim)fHisX)-cyt c. The line (set at an ET rate of 3 x 1012s-l for the one-bond limit) represents the best fit to the data (slope = 0.71 A-1).

Related experiments aimed at understanding the role that protein structure plays in modulating ET rates have been conducted using a Ru-labeled cyt 4 complex [13*]. The sulfhydryl group of the Thr65Cys cyt 6, mutant was labeled with [4-(bromomethyl)-4’. methylbipyridine](bisbipyridine)Ru2+ to form Ru-65-cyt 4. The forward and backward ET rates between the Ru complex and the heme group are summarized in Table 3. The excited-state ET rate from *RuZ+ to Fe3+-cyt 65 is 1.4 X 1O7s- l (a minor component of the kinetics, associated with a ritte of 3 x 106s- 1,was also observed) and kt back from Fez+-cyt 65 to Ru3+ is 6 X 106~t. Fitting these data to the Marcus relationship (Eqn 2) yielded a 3. ET parameters

for Ru-65-cyt

Reaction ‘Ru2+

b,

1.4 x

cyt 6 ET reactions just In addition to the ruthenium discussed, Willie et al. [ 13.1 have also explored interprotein ET between cyt 6 and its natural redox part ner cyt c. Biexponential kinetics were observed (80%: 4 X 105s-l; 20%: 3 X lo4 s-‘l>, illustrating the potential complications that arise from protein motion on the time scale of these ET reactions. In this relatively well defined complex, structural modeling and energy transfer experiments generated estimates of the heme-heme distances ranging from 8.5 to 12.5A.. Uncertainties in the rates and distances for interprotein ET make it difficult to estimate the theoretical parameters of ET.

b,.

-AC

k,r (s-1) + Fe3 + -cyt

(s-1)

107

0.82

6.0 x 106

1.28

Data

from

A feV)

1.0 Fe?+ -cyt aCalculated

b, + Ru3 + by us in this

review.

Farid, Moser and Dutton

We have also measured, using the model of Moser et a[. [6**], the edge-to-edge distance, &, from the ruthenium complex to the heme delined by the authors’ rules [6**], i.e. between the Ru bipyridine ligands and the heme Fe. We find that the distance ranges from 14.5 to 17A for all allowable conformations of Cys65, with 16.6A beingthe distance associated with the lowest energy structure. Given that the heme group and Ru complex are connected via a covalent bridge, the estimated k,, of 1.8 x 107 s-1 would be expected to fit on the line in Fig. l(b) that accommodates rates for covalent model complexes C/3= 0.7A-1). The line predicts indeed a k,,, of 2.4 x 10’ s-l for the measured 4, whereas the line of Fig. l(a) predicts a rate two orders of magnitude slower (1.3 x 105 s-l >. These results suggest that a covalently linked donor-acceptor molecule incorporated within a protein exhibits electronic coupling similar to bridged complexes in a fluid solvent.

The distances used in the above analyses differ from the definitions used by Moser ef u[. [6-l, which, if applied to the above data, partially account for the deviations of His72 and His33 from the best-fit line of Fig. l(a), as only in these proteins are the Ru complexes positioned such that the axial ligands of the heme intercept the path between donor and acceptor. For example, the His72 R, is 12.6A, measured from the Fe atom of the heme group to the edge of the protein imidazole ring of the Ru complex, and excluding the heme axial histidine ligand. This moves the His72 point to within a factor of 40 from the Dutton line in Fig. l(a) [see Table 21. The measurements demonstrate some of the uncertainties that still remain in the definition of distance, and highlight the need for proper evaluation of donor-acceptor boundaries representing the ET event.

Table

in proteins

k,, of 1.8 x 107 s- * (ET parameters: V, = 0.26cm-l, L = 1.0 eV>. The Ru(bpyj3 complex was shown to be. covalently linked to the heme-Fe via His63-Se&Cys65 connectivity. As the authors point out [ 13*], this pathway contains 12 covalent bonds, which we have calculated, using the Beratan and Onuchi approach, to have an effective o-tunnelling length, 01, of 16.8h We have compared the k,, and associated 01 for this reaction with the results of Gray and coworkers [ lO**,ll*], which is appropriate because the donors and acceptors are nearly identical in the two systems (i.e. the pre-exponential teml, A, in Eqn 6 have similar values). Accordingly, the best-fit line of Fig. 3 predicts a rate of 4.0 x 107s- * for the given 01, which is only about twofold greater than the experimental k,,. This suggests that the pathway model of Beratan and Onuchic represents adequately the protein medium between electron donor and acceptor and accounts for the observed rates,

log km (s-V

5

transfer

reproduced

[I?,*].

V,

(cm-7

k max (s-9

R (A)

0.26

1.8 x

12.4

107

01”

(A)

16.8

229

230

Theory

and simulation

,

Experiments aimed at understanding the role that protein plays in modulating ET rates have also been conducted in several closely related species of the blue-copper protein azurin [ 14.1. ET from the disulfide bridge radical anion, RSSR=- (produced by pulse radiolysis), to the Cu(II> center was studied. In the three azurins studied, the disuffide bridge (~ys3-cys26) is found at one end of the 8-barrel-structured protein. In Pseudomonas aerug inosa azurin, for which the three-dimensional struct)lre has been determined, the donor-acceptor edge-to-edge distance, %, is 25.24 as measured from the Cu atom. to the S atom of Cys26. The only significant dilference in the sequences is the identity of the residue at position 48, which is presumed to intercept the path between the Cu center and the disuffide bridge; i? aeruginasa azurin contains a Trp48 whereas P. jluorescem and Alculigenes faecalis azurins contain Leu48 and Va148, respectively. The values for kt (see Table 4), after accounting for possible variations in driving force, reorganization energy, and distance, indicate that the electronic coupling for P. aeruginasa azurin is about threefold greater than for the other two azurlns. We have confirmed this difference by calculating the values of k,, for all three species using the Marcus relationship in Eqn 2 (see Table 4) and using the driving forces estimated by the authors from the ground-state electrochemical properties of the Cu’ center and the disuffide group, and their estimate of h (1.2eV) [14*,15]. A possible explanation for the observed rate variations was attributed to the effect of the indole side chain at Trp48 on the electronic coupling. To confinn this idea, Farver and Pecht [14*,15] have applied the Beratan and Onuchic pathway analysis to the P, aeruginasa azurin structure. The analysis revealed a pathway through Trp48 that contained 23 covalent bonds, two hydrogen bonds, and one 3.58 through-space jump. Although a specific pathway analysis of the two other species was not conducted, the implication is that Va148 or Leu48 side chains cannot contribute as effectively as Trp48 to the pathway coupling between donor and acceptor. From the results, the authors suggest that aromatic residues may be selected by nature to increase considerably the electronic coupling in specific regions of this and other ET proteins. In addressing this issue, we lind that the effect on ET rates (up to a factor of 3 for calculated k,, values) are well within the scatter of the data in Fig. 1. Yet, upon a more

Table 4: ET parameters Species Pseudomonas Pseudomonas Alcaligenes aCalculated reproduced

for Ru-6.5cyt Residue

aeruginosa fluorescens laecalis by US in this review. from WI.

detailed examination of the protein structure, it was evldent to us that the side chain of residue 48 only grazes the straight-line path between the Cu center and the disulfide bridge. Also, considering the o-tunneling pathway in P. aeruginom and in modeled structures of P. Ji’uorescens and A. faecalis azurins, we lind that the best electronic coupling occurs through a pathway that does not involve the residue at position 48 for any species. In contradiction with the author’s calculations, the single best pathway for all species of azurin studied started from the S atom of Cys3, continued up the backbone of a g-strand (Gln8-Ile7-Aspb-ValSSer4) to Gly9, then to the Cu his&line ligand (His46) via a through-space jump of 3.78A and onto the Cu center (01 = 46.5A, -4t! shorter than the pathway of Farver and Pecht). Therefore, with the possible exception of l? J’uorescens, which contains Leu48 and which might exist in a conformation that yields a larger coupling pathway (up to a facror of 2), we find that the pathway analysis as implemented predicts that all three species of azurin should have identical maximum rates. We make no attempt to compare these values of 01 and associated k,, values with those of reactions in Ru-modified cyt c and cyt 6s because the values for the pre-exponential A in Eqn 6 are most certainly different because of the disparate nature of the donors and acceptors. The measured and estimated values for % and k,, can however be compared with the best-fit line of Fig. l(a); an & of 25.2A and k,, values of 100 and 300 s-1 place the data more than two orders of magnitude above the Dutton line. As we suggested earlier, this enhanced coupling could originate from the highly covalent nature of the path between the Cu center and the disuffide bridge. As far as the differences in k,, among the species of azurin are concerned, we note that the threefold variation in rates can be accommodated by % fluctuations of as little as 0.8A, assuming an average p of 1.4A-1. It is reasonable to expect that such small differences in distance across the 25A donoracceptor separation would arise from structural variations between the species of azurin, even allowing for the reported high 70% sequence homology. Although structures are not available for all of the species of azunn studied, we lind that both the % distance and the pathway analysis cannot unambiguously account for the observed ET rates.

b,. 48

Trp Leu Val &stimated

ket (s-1)

- AC’ teV)

71 (eV)

V, (cm-11

x 10-X

44*7

0.71

1.2

1.2

22 f3

0.76

1.2

6.5 X IO-'

11 f2

0.68

1.2 ,

6.5 X IO-4

from

modeled

structures

based

on X-ray

structure

k,,,a

(s-1)

Ra

(A)

da (A)

310

25.2

46.5

100 100

x.2" x..@

46.5" 46.5"

of P. aeruginosa

azurin.

Data

Electron

Electron

transfer

in synthetic

polypeptides

The protein systems described above have yielded valuable insights into the roIe that the protein medium plays in controlling ET rates in biological and semibiological systems. The complexities inherent in these systems have however led some groups to investigate intramolecular ET reactions in synthetic polypeptlde structures. Synthetic systems potentially offer the unique advantage of nearly unlimited flexibility in modifying the environment between and around donor and acceptor molecules, thereby making it possible, in principle, to modulate almost freely the distance, driving force, reorganization energy and the nature of the intervening medium. But because, in part, of the relatively small size of the synthetic polypeptides studied, the folded structures can accommodate many low-energy conformations, making assessment of the above ET parameters difficult. Common to all the systems described below is the dependence on modeled structures as the only source of structural information, including donor-acceptor distances. An example of such a polypeptide system aimed at addressing some of the issues presented in this review is a synthetic monomeric a-helical peptide in which ET from a tryptophan radical (Trp*) to a tyrosine (TyrOH) was studied [16-l. The donor, Trp*, is generated by the reaction of Eqn 9, and ET to TyrOH is represented by Eqn 10: N; + TrpH - (helix) -TyrOH (9)

transfer

in proteins

Farid, Moser and Dutton

231

In this case, we have not attempted to model this peptide, leaving I’$ values and thus p undetermined. Because. - AG” values were not reported, assessment of k,, in these peptides is not possible; however, the calculated p values are still surprisingly low, such that, aside from structural uncertainties, an explanation is not available. A similar study conducted in a-helical polypeptides containing a single pair of L-~(dimethylamino)phenyl%nlne and t-1-pyrenylalanine residues has also been reported, [18*]. The distance and orientation of the (dimethy1amino)phenyl (donor) and pyrenyl (acceptor) groups were varied by inserting different numbers (m) of Lalanine units between the artificial aromatic amino acids. The shortest edge-to-edge distance between donor and acceptor groups varied in a non-linear fashion from 5.4 to 9.4A as m was altered from 0 to 2. These distances and the rates of photoinduced ET from the donor to the triplet excited state of the acceptor are given in Table 5. Although the data is very limited, 8 in this helical system was estimated to be 0.9A-1, a value similar to the p of 0.7A-1 obtained from the investigation of covalently linked donor-acceptor molecules presented in Fig. l(b). This suggests that, unlike the previous example where j3 was unusually low, these helices retained their, predicted conformation in solution, leading to ET mediated by an effectively covalent bridge between the donor and acceptor molecules. It is therefore possible that in a helical structure, the observed trend in rates would not continue if m was increased to 3 and beyond, as noncovalent interactions would most likely begin to play a role in determining the pathway coupling between the redox centers.

Trp’ - (helix) - TyrOH + NT + H+ Table

Trp’ -(helix) - TyrOH ZTrpH-(helix)-TyIo’(10) An attempt to modulate the donor-acceptor distances was made by varying the number of residues separating the tyrosine and ttyptophan, which were contained within the a-helical structure [ 16.1. In a helix where the intervening residues were alanine, lysine, glutamate and a-methylalanine (2aminoisobutryic acid; Aib), the exponential coeficient of decay of ET rates per residue, b,, was found to be 0.2. This decay parameter is invoked instead. of the usual p because of the lack of distance information. We have measured the distances in structures generated by computer modeling whereby a plot of In&t) versus 4 was generated, yielding a line associated with a g of O.l3A-1 which correlated reasonably well with the data (R = 0.92). (We assumed ideal ahelixgeometry: += -58fl”and JI= -47&l”, and X-angles set at the most frequently observed values given in [17]. The & distances measured in the modeled structures and the observed rate (k,J are as Follows: for three intervening residues, 4.4% 1.7 x 104 s- 1; for six, 8.1, 4.7 x 103; for seven, 11.1, 4.7 X 103; for eight, X4.2, 4.2 X 103; for 11, 17.3, 2.7 x 103; and for 13, 18.2, 1.8 X 103.) Interestingly, in a helix where the intervening residues were proline, b, had a much larger value of 0.6.

5. ET rates for donor-Ala,-acceptor

m

(A)

R,

a-helical

5.4

1.9 x 107

1

9.4

6.6 x 105

2

5.5

2.1 x 107

reproduced

from

I

ket (s-~)

0

Data

peptide.

L17.1.

.

An issue not yet considered in this review, the role that donor-acceptor orientation plays in controlling ET rates, was also addressed in this system. Although the rates and distances for the m = 0 and m = 2 helices were similar, in the Former, the donor and acceptor ring structures took on a presumed edge-on conformation relative to one another, whereas in the latter, a face-on.conformation was assumed to prevail. Although three-dimensional structures are not available, it appears from this work that the relative orientation of the donor and acceptor does not play a significant role in controlling ET rates. A similar conclusion was also reached in the investigation that led to the data presented in Fig. l(b) [6**]. Many complications that arise in the current systems prevent unambiguous assessment of the role of distance and intervening medium in controlling ET rates. It is clear

232

Theory

and simulation

however that the potential exists for synthetic polypeptide systems that ark structurally well defined to address the issues presented in this review, by allowing almost unconstrained control of distance and intervening medium.

Conclusion

I In considering the factors that govern ET reactions, we have adopted a biological point of view which emphasizes natural selection and engineering of biological systems. The large variety of intra- and interprotein ET systems now being described is beginning to provide meaningful conclusions. The directional specificity of ET is a principle biological engineering concern, both for elementary (e.g. those involving cyt c) and the more elaborate energy conversion proteins (e.g. photosynthetic reaction centers, cyt c oxidase or the cyt bcI complex). Thbre is no doubt about the essential role ,of distance, driving force and reorganization energy in these processes [6-,191. These three principal factors appear indeed to be sufficient to engineer the demanding ET networks of energy conversion [19]. Whether or not ET in natu’ral systems receives further guidance from designed stmc&al elements of protein medium is less clear. Test examples of non-physiological ET reactions between hemes and ruthenium coordination complexes, occurring through unusual regions of proteins picked out as candidates for hindered or promoted transfer, support the view that sign&ant control of ET rates from the protein structures is feasible. Yet our observations with reaction centers demonstrate that pathways selectively promoting physiologically useful over wasteful ET reactions are absent. It remains to be seen whether or not an expanded search of physiological ET systems will show that natural selection has acted on the medium between redox centers to control intraprotein ET.

Acknowledgements This work was supported by National Institutes of Health grant GM41048. RS Farid acknowledges an NIH postdoctoral fellowship.

References

an’d recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1.

2.

Mucus RA: On the Theory of Oxidation-Reduction Reactions Involving Electron Transfer. I. J C&em Phja 1956, 24:96@78. Gout, IR, MOODY R, FARID S: Electron-Transfer Reactions in the Marcus Inverted Region: Differences in Solvatlon and Electronic Coupling between Excited Charge-Transfer Complexes and Germinate Radical Ion Pairs. J Am Cbem Sot 1988, 110:7242-7244.

3.

.

CID% GL, CALCA~RRALT, GREENNJ, PENFIELDKW, MIIUR JR: Distance, Stereoelectronic Effects, and the Marcus Inverted Region in Intramolecular Electron Transfer in Organic Radical Anions. J PLys Cbem 1986, 90:3673-3683. VG, Doco~ma RR: Teoriya BezizluchatelnIkh Electronnikh Perekhodov Mezhdu Ion&i Rastvorakh. Dokl Akad Nauk SSSR1959, 124:123-126.

4.

UUCH

5.

JORTNER

6. ..

MOSER

7.

MILLERJR, BEI’IZ JV, HUDDLESTONRK: Effect of Free Energy on Rates of Electron Transfer between Molecules. J Am UJCIII ~gc 1984, 106:jO57-j068.

J: Temperature Dependent Activation Energy for Electron Transfer between Biological Molecules. J C%em PLga 1976, h4860-4867.

CC, KEw JM, WARNC~ K, FARED RS, DUITON PL Nature of Biological Electron Transfer. Nalrcre 1992, 355:796-802. An analysis of intraprotein ET is developed from ET measurements in both biological and chemical systems. From data spanning 2OA in distance and 12 orders of magnitude in rare, it is shown that proteins, like an organic glass, present a uniform electronic barrier to electron tunneling.

BERATANDN, B!xrs JN, ONUCHICJN: Protein Electron Transfer Rates Set by the Bridging Secondary and Tertiary Structure. Science 1991, 252:1285-1288. It is predicted that the distance dependence of ET in proteins is controlled by the protein’s ‘structural motif. The caiculations use a tunneling pathway model which is applied to several cyrochromes. allowing the generation of color maps showing ‘hot’ and ‘cold’ electronic coupling regions of the protein. The model is presented as a potentially powerful tool towards the design of molecular daices which can achieve energetic eficiency by utilizing tunneling pathways. 8.

..

9. .

BERATANDN, BEITS JN, ONUCHIC JN: Tunneling Pathway and Redox-State-Dependent Electronic Coupling at Nearly Fixed Distance in Electron-Transfer Proteins J Pkys Chem 1992, 96:2852-2855. It is shown that the application of a pathway analysis that considers covalent, hydrogen-bonded and through-space contacts between donor and acceptor in proteins can lead to testable predictions concerning ET rates at short ( < 5 A) distances in a-helix versus P-sheet strucrures, and in oxidized versus reduced or ligated versus deligated metalloproteins. 10. ..

WUT~KE DS, BJERRUMMJ, WINKLERJR, GMY HB: ElectronTunneling Pathways in Cytochrome c Science 1992, 256:1007-1009. This paper presents ET data for the reactions between Ru bipyridine complexes attached fo surface histidines and the heme group of cyt c. Convincing arguments are made for the validity of the model presented in [8”,9’] in explaining the unusually slow rates when viewed in terms of the model presented in lb**]. Through-space and hydrogen-bond interactions in the pathways connecting donor and acceptor appear to attenuate the electronic coupling. 11. .

Wunw DS, BJ~RRUMMJ, CHANG I, WINKLERJR, GRAY HB: Electron Tunneling in Ruthenium-Modified Cytochrome c Biocbim Biopbys Actu 1992, 1101:16&%170. Electron couplings are calculated for intramolecular ET in four ruthenium-modtied cyts c The rates are successfully correlated with the lengths of o-tunneling pathways comprising covalent bonds, hydrogen bonds, and through-space jumps between the Ru complex and the heme group. 12.

CHANGI, GRAYHB, WUUKLER JR High-DdvIng-Force Electron Transfer In MetaIIoproteins: Intramolecular Oxidation of Fercocytochrome c by Ru(2,2’-bpy)2(im)(Hiss-33)3+. J Am Chem Sot 1991, 113:70567057.

13. , WIIUEA, STAYTONPS, SUGARSG, Duv B, M1wa-r F: Ge. netic Engineering of Redox Donor Sitea Measurement of Intracomplex Electron Transfer between Ruthenium65+tochrome 4 and Cytochrome c Biocbemisfry 1992, 32~7237-7242. The authors present data on both intra- and interprotein ET. In the former. the donor and acceptorare covalendy bridged within the protein

Electron complex. The potential complications arising from the latter study are clearly presented. 14.

FARVER0, PECM I: Long Range intramolecular Electron Transfer ln Axurins / Am C&em Sot 1992, 1145764-5767. b data are presented for the reaction between the disulfide bridge and Cu(II> center in three species of azurin. Analysis of the medium between donor and acceptor reveals a tryptophan residue in the pathway for one of the species, suggesting that the observed faster rate compared to two other species is linked to the aromatic side chain. Potential factors that might lead to differences in ET rates are discussed thoroughly, then dismissed tentatively, leaving the protein medium as the controlling factor of ET rates. 15.

16. .

FNWER 0, PECHT I: Long-Range Intramolecular Electron Transfer in Axurins. Proc Null Acad Sci Cl S A 1989, 86:6968-6972.

bE H, FARACGI M, KL4PPER MH: Long Range Electron Transfer Along an a-Helix. Biarhim Biophvs Acka 1992, 1159286294. A detailed study demonstrating the usefulness and some of the complications of using synthetic a-helical polypeptides in addressing the roles played by medium and distance in interprotein ET. Theoretical

transfer

in proteins

Farid, Moser

and Dutton

treatment of this and related systems will certainly lead to a better understanding of these issues. 17. PONDER J, Rtcn~aos FM: Tertiary Templates for Protein: Use of Packing Criteria in the Enumeration of Allowed Sequences for Different Structures. J Mol Biol 1987, r 193:775-791. 18. .

Y, Stsmo M, lhiwts~l X Photoinduced Electron Transfer on a Single a-Helical Polypeptide Chain. Evidence of a Through-Space Mechanism J P&s Cbem 1991, 95:3847-3851. A study of ET between redox groups incorporated into a synthetic ahelical SttUCNre. The distance between the redox groups is varied by insertion of one to three intervening residues. The suggestion is made that ET occurs by a through-space mechanism, although the possibility of long-range ET through the polypeptide chain cannot be excluded. 19. MOSER CC, DLTI’ON PL Engineering Protein Structure for Electron Transfer Function in Photosynthetic Reaction Centers. Biocbim Eiophvs Acka 1992, 1101:171-176. INAI

RSFarid, CC Moser and PL Dutton. B501 Richards Building, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104.6089, USA.

233