Experimental Approaches to Studying Biological Electron Transfer R M A. Scott School of Chemical Sciences, University of Illinois, Urbana, IL 61801
A. Grant Mauk Department of Biochemistry. University of British Columbia, Vancouver, BC V6T 1W5, Canada Harry B. Gray Arthur Amos Noyes Laboratory, California Institute of Technology. Pasadena, CA 91 125
Electron transfer reactions are central to many of the metabolic processes necessary for the survival of all organisms. Electron transport chains function as a series of consecutive electron transfer reactions between pairs of proteins which have evolved to couple efficiently the exergonic electron transfer events to energy conservation. Typically, the electrons are transferred between metal sites or organic prosthetic groups that are spatially arranged within a single protein or a complex of proteins such that the electron must traverse a considerable amount of intervening polypeptide. The observation of significant rates of electron transfer between sites separated by perhaps 20-30 A has raised the possibility that electron tunneling is the operative mechanism in these cases. The obvious importance of electron transfer in biological systems and the possibility that tunneling occurs in some biological electron transfers have contributed to the continuine interest in theoretical and exnerimental studies of these reactions. Perhaps due to the apparent simolicitv of the electron transfer event. theoretical predictions or expected characteristics of biological electron transfer reactions have often preceded the necessarv . exoeri. mental measurements. Recause d this rhronoloyy, most rurrent experimental studies of biolorical elertron transfw are designid to test various theoretical predictions. I t is the purpose of this article to describe some of these experimental studies. Biological electron transfer reactions may he naturally divided into two types: intermolecular electron transfer between sites residing in different protein molecules; and intramolecular electron transfer between fixed sites within a single protein molecule. When discussing electron transfer reactions between proteins that form Dart of a membranebound complex, the differences may become blurred. For example, consider the terminal portion of the mitochondria1 respiratory chain. Intermolecular electron transfer occurs between cytochrome bc, (Complex 111, cytochrome c reductase) and the soluble cytochrome c and between cytochrome c and cytochrome aa3 (Complex IV, cytochrome c oxidase), whereas the electron transfer from heme a (and CUA)to heme a3 (and Cue) within cytochrome an3 is an example of intramolecular electron transfer. If cvtochrome c and cvtochrome e oxidase exist as a protein-protein complex for a sianificant amount of time. then the heme c to heme a elecrron transfer may even be considered as an "intramolerular" (actualls intra-comr)lexJ reaction. In the terminolom -.of inorganic-electron trsnsfer, such a complex is known as a precursor comolex. This conrept gives us a unified framework for disrussing both types of biological elertron transfer. Equation 11) representsa general electron transfer reaction involving an electron donor (D) and an electron acceptor (A):
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For intermolecular electron transfers, K, and K, are formation constants for the precursor complex (AIID) and the successor comdex (A-D+). res~ectivelv.The actual electron transfer eieut consists of conversion of the precursor to the successor complex and has a rate constant, k,,. We need only consider t h i s reaction when discussing i&&olecular electron transfer. The rate of intramolecular electron transfer reactions will depend only on factors which influence ket. However, intermolecular electron transfer rates will alsodepend on factors affecting the formation of the precursor complex. Thus, diffusion rates (e.g., three-dimensional diffusion in solution or two-dimensional diffusion in a membrane) and electrostatic effects will contribute to the overall electron transfer rate in intermolecular hut not in intramolecular reactions. In some cases. these effects mav be separated kineticallv - (i.e., . . when saturation kinetics are-observed), hut often this separation must depend on overly simplistic calculations of the electrostatic effects. Since the goal of most experimental studies of biological electron transfer is to define the effects of the molecular and electronic structures of the acceptor and donor on k,,, studies of intramolecular electron transfer have the advantage of avoiding the need to make such a separation. Considering a protein molecule with two fixed (acceptor/ donor) sites between which intramolecular electron transfer occurs, the characteristics that will affect the magnitude of k,t may be delineated: (1)the intersite separation distance; (2) the drivinp force for the reaction (i.e.. the difference between the r&uctiun potentials of the acceptor and donor); (31the nature of the interveninr medium; and (4) the relative orientation between the (presumably anisotropic) acceptor and donor sites. Studies designed to probe each of these questions are the subject of this article. Fundamental Theory. The details of the actual electron transfer step (k., in eqn. (1))are more amenable to theoretical study than are the interactions involved in precursor complex formation. The important concepts are most easily visualized hy examining the potential energy surfaces in Figures 1 and 2. In Figure 1, the reactants (precursor complex) are characterized by the AI/D curve and the products (successor complex) by the A-IID+ curve. These curves represent the potential energy of the system as a function of some ill-defined nuclear coordinate. There will always be some change in equilibrium nuclear positions upon electron transfer, and the nuclear coordinate used in Figures 1and 2
represents the combination of nuclear positions which are changed in going from reactants a t equilibrium to products at equilibrium. If A and D are separated by an infinite distance, electron transfer cannot take place and the potential energy curves of Figure 1 would follow the dashed lines. The system would remain on the AllD curve and never move to the A-IID+ curve. However, in the true AIID precursor complex (or in a svstem with two fixed sites a t reasonable separation), sites A a i d D can communicate electronically andthis causes mixing and splitting of the energy surfaces in the crossing-point region as illustrated by the solid lines of Figure 1.The stronger the electronic interaction, the greater the splitting (AE) between the upper and lower curves and the higher the probability for conversion of AllD A-IID+ once the system reaches the nuclear coordinates of the crossing point (R,). In the limit of strong electronic interaction, AE becomes so laree that the u m e r enerev surface mav be innored and the &;em has unit'prohahili~; for movin~from'theA D curve to the A- 1)' curve unrr ut R.,,. This situation is referred tu as an adiabatic electron transGr. In systems for which the electronic interaction between A and D is weak, AE (Fig. 1) is small and there is still some probahility that the system will remain on the AIID curve when passing through the crossing-point (i.e., that it will follow the dashed lines in Fig. 1). When the probability of reaction is less than unity for systems reaching the crossing
-
Nuclear Coord~note,R Figure 1. Potentlal energy curves representing a general electron transfer reaction, The abscissa is an illdefined nuclear cwrdinale representing a combination of metal-iioand vibrational modes. The len curve reorasents the potemla energy ol the reactants (precursor complex A Dl and tne r ght curve me polenl8a. energy al the produns wccessor complex 4 - 0.1 LE' s the activatcon banier lor the electron transfer reaction and AEis the energy gap between the mixed and split potential energy curves, reflectingthe amount of electronic interaction between the A and D sites.
Figure 2. Potentialenergy curves (as in Fig. 1)showing the effect of varyingthe drivlng farce on,the activation barrier for electron transfer. (a) With small driving forces ( A P ) , the activation barrier (AE') decreases when A P becomes more negative. (b) The transition point into the "invwted region." (e) In the "inverted region." more negative A P causes en increase in A F .
point, the electron transfer is called nonadiahatic. Although adiabatic behavior is dominant in normal chemical reactions (including small molecule electron transfer), biological electron transfer is most likely dominated by nonadiabatic behavior. The rates of nonadiabatic electron transfer reactions are dependent on the size of the splitting (AE) between the levels a t the crossing point:
As indicated by Figure 1, the nuclear coordinate must distort to a configuration common to both reactant and product potential surfaces (i.e., the crossing point) before electron transfer can take place. Normal vibrational motion is responsible for this distortion and if the system has enough vibrational (thermal) energy to overcome the harrier (a*), then electron transfer occurs by an activated process. At lower temoeratures. where the svstem does not have enough thermal energy to overcome the harrier, electron transfer may still occur by (nuclear) tunneling through the harrier. This is possible due to overlap of the tails of the vibrational wavefnnctions for all AIID and A-IIDf confieurations in the crossing-point region below the barrier. ~ t v e r ~ low temperatures, only the lowest vibrational level of the AllD potential surfaces will be populated and the electron transfer (tunneling) rate becomes temperature-independent. From this discussion, it should he clear that properties of the svstem that affect the size of AE will affect the rate of nonadiahatic (e.g., biological) electron transfer. Since AE is directly proportional to the mixing matrix element which is controlled by the amount of overlap of the A and D electronic wavefunctions, anything that affects this overlap will affect the electron transfer rate. For example, the distance (r) between A and D will affect this overlap. Hopfield's vibronically-coupled electron tunneling theory predicts a distance dependence of the electron transfer rate as
since a t reasonably large separation distances the electronic wavefunction overlap between A and D is confined to approximately exponential tails. If A andlor D are nonspherically symmetric sites, their relative orientation will also influence the amount of overlap between the accentor and donor electronic wavefunctious. affecting the electron transfer rate. If there exist intervening molecules (amino acid side chains) with electronic levels at accessible energies, their presence will increase the effective overlap between acceDtor and donor electronic wavefunctions. These consider&ions also lead to the question: How does one define the extent and nature of the acceptor and donor electronic wavefunctions? In principal, one must consider liaands that have molecular orbitals mixed in with the metal d orbitals as representing extensions of the acceptor1 donor site. Thus in measuring distances between sites with porphyrin-type coordination or histidine imidazole ligands, one should probably consider these ligands as defining the outer fringes of the acceptorldonor electronic wavefunctions. Detailed theoretical studies of these effects are lackinn. Another property of the system that affects the electron transfer rate is the drivine force for the reaction. Fiaure 2 illustrates this in terms o r potential energy curves. As was first pointed out by Marcus, the activation energy required for electron transfer (AE*) should initially decrease with increasing driving force (AEo),but AE* goes through a minimum (Fig. 2b) and then increases with increasing driving force in what is referred to as the "inverted region" of electron transfer (Fig. 2c). For highly exergonic electron transfer reactions in which there is little rearrangement of nuclear positions a t the acceptor/donor sites during electron transVolume 62 Number 11 November 1985
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fer, the minima of the two potential energy surfaces are displaced very little horizontally from one another and there may be no crossing point (at any accessible energy). In such cases, electron transfer must involve nuclear tunneling at any temperature. Experimental Approaches
Studies of Bimolecular Electron Transfer Reactions Electrostatic Effects. In studying electron transfer reactions (biological or chemical) between two freely diffusing reactants, one would like to he able to separate effects of the medium on precursor complex formation from effects related to the actual electron transfer steo. A maior of the . nortion . medium effects in aqueous solution are associated with the charges of the reactants. The magnitude of these electrostatic effects depends on the ionic content of the aqueous medium. and, ionic strength deoeudences of electron transfer rate constants can yield-informatiou about the electrostatic interactions between the two reactants. Such data are straightforward (though tedious) t o measure so that much of the research effort expended has involved attempts to devise a theory to predict the ionic strength dependences observed. The simplest method of treating the electrostatic interaction between a metalloprotein and a small molecule redox agent is to assume that both metalloorotein and small molec;le are uniformly charged hard spheres and to use DehyeHuckel theory to calculate the rate as a function of ionic strength. This approach was developed by Wherland and Gray (1) and was used to correct bimolecnlar rate constants for electrostatic effects so that relative Marcus theory could he used to correlate rates from reactions with differently charged reactants. This simple theory does not succeed in quantitatively predicting observed ionic strength dependences, presumably due to the typically asymmetric charge distribution on the metalloprotein. The imnortance of considerine this asvmmetric charee distribution is demonstrated by studies involving chemical modification of specific charged amino acid side chains on the metalloprotein reactant. The best-studied example involves specific lvsine modification of cvtochrome c and its electron-transfe; reactions with some physiological partners (2-11) and some nonphysiological redox agents (12-14). Modification of the r-amino group of a lysine residue by several different chemical reagents results either in neutralization of the positive charge or replacement by a negativelycharged group, thus altering in a known specific way the charee distrihution of cvtochrome c. The eeneral results of these studies show that modification of Gsines in a small well-defined reeion surroundine- the exposed heme edge - of cytochrome c affect electron transfer rates, hut lysines outside of this reeion have no effect. Thus, use of the total protein chargeand the overall dimensions of the protein to calculate a charge densitv on a sphere would not he expected to yield re~iable~redictibns of electrostatic effects. In order to do a proper theoretical treatment of these eff~cts,one nust con~siderthe actual charge distribution on the metalloprotein. A simple approach is to treat the small molecule-prowin interaction as a point (or small sph~rical) charge interacting in a Coulomh fashion with a specific 3. dimensional arrav of uoint rharees orotein). This ao.ithe . proach has been &ed by Millett and coworkers in a study of the oxidation of ferrocytochrome c by Fe(CN)e3- (12). In this study, the rate variation with different specifically-modified cytochromes c was nsed to localize the site of attack of Fe(CN)e3- on cytochrome c. Unfortunately, no ionic atrength-dependent rates were measured to test whether this appn~achis better than simple Debye-Hiwkel theory in that regard. This approach considers only monopole-monopole interactions, yet the asymmetric charge distribution of cyto934
Journal of Chemical Education
chrome c is known to give rise to a large dipole moment. Other theoretical studies have included dipole-monopole interactions as well (15-19) and for cvtochrome c. the oositive end of the dipole is shown to directly at the reactive region of the surface near the exposed heme edee (17). I t has been suggested that the dipole moment of cytochrome c is responsible for proper orientation of the protein in complexes with its physiological partners and that this is an example of biological specificity (17). Precursor Complexes. Considerable interest has developed in recent years in demonstrating the occurrence of kineticallv detectable orecursor comolexes in electron transfer reactions between proteins and small molecule reagents. Historicallv, the iron hexacvanide-cvtochrome c reactions were the f i k t to he considerid in these terms in studies that involved both NMR (20) and equilibrium dialysis (21). Detection of this interaction in rapid-mixing kinetics studies, however, has been problematic. The oxidation of ferrocytochrome c by ferricyanide is quite fast, so precursor complex formation is difficult to detect and relatively few studies of this reaction have been reported. The apparent rate saturation initially reported for the reduction of ferricytochrome c hv ferrocvanide a t low ionic streneth (22) was suhse, quently recognized as arising from substantial contribution of the back-reaction to the absorbance change monitored (23,24). Nevertheless, the involvement of a precursor complex in the reaction mechanism is generally accepted (23, 24) - -, Sykes and co-workers have reported limiting or saturation kinetics in the reactions between a variety of type 1 copper proteins or iron-sulfur proteins and small molecule reagents (2530). Although in many of these cases the dependence of kobs on reagent concentration is more accurately described as nonlinear rather than saturatine. true rate saturation has been observed at least in the oxization of HiPIP with Co(4, 7-DPSohen)q3- (4. 7-DPSnhen 1 4. 7-Diohenvlsulfonate-1. 10-phenanthroline) (29) Hnd the oxidation bf ferredoxid with (NH8)&oNHzCo(NH3)s5+, Co(N03),$+, or Pt(NH3)$+ (30). The interactions between protein and reagent that have been interpreted by Sykes and co-workers as arising from precursor complex formation are electrostatic in nature and are favored in reactions between highly and oppositely charged reactants. Limiting kinetics had also been reported previously for the oxidation of azurin by Co(4, 7D P S o h e n W (31). hut no detailed interoretation of this result was offered a t that time. Interestingly , no such reagent-protein interactions have been observed (24) in any of the multitude of reactions reported for oxidation or reduction of cytochrome c, which is highly cationic. One report on cytochrome b5, which is highly anionic, does indicate limiting kinetlcs in its reactions with several inorganic oxidants (32). To characterize the protein binding sites of those redox reagents that demonstrate limiting kinetics in their reactions with metalloproteins, Sykes a i d co-workers have nsed redox-inert coordination camplexes with homologous structures as competitive inhibitors to map out hinding domains for various types of reagents on protein surfaces (e.g., ref. 33-36), NMR spectroscopy has provided an alternative approach to analvsis of reaaent-protein interaction. For example, the - . interaction of iron hexacyanides and related complexes with cytochrome c has been studied by NMR to evaluate both the stoichiometry and location of hinding (37-39). From this work i t appears that there are a t least two binding sites for iron hexacvanides on cvtochrome c with association constants 5 3 % lo2M-' in chloride-containing media ( p = 0.12 M) that are independent of protein oxidation state. These results are consistent with those obtained from some kinetics studies (24. 40). but discre~ancieswith other kinetics results may be ;elated at least in part t o specific ion effects such as chloride hinding to cytochrome c (39). Complete rationalization of the large number of kinetics studies re-
ported (4145) for these reactions remains as a persistent challenge. In studies examining the interactions between other pairs of small redox reaeents and metallonroteins. the dominant strategy has been to use the chromi&n analog of the iron or cobalt comnlex found to demonstrate kineticallv detectable precursor complex formation as a redox-inert paramagnetic probe to induce line broadenine in nrotein resonances that are attributable to residues involvedin metal complex binding. In this way, the binding sites of [Fe(CN)s]3- and [Co(phen)13+on plastocyanin have been studied (4648) as has the binding site of [Fe(CN)d3- on Pseudomonas azurin (49). In the latter case, bindingsite between Lys-85 and Lys-92 has been proposed. A similar study of the interaction of Fe(EDTA)- with horse heart cvtochrome c has detected the existence of two binding sitesWforthe complex on opposite sides of the heme crevice that would result in Fe-Fe seoarations of 12 or 13 A (50). An affinity approach to identification of redox reagent hinding sites on metalloproteins has been developed by Pecht (5153) based in part on earlier observations by Kowalsky (54) and Fleischer and co-workers (55) on the reduction of ferricytochrome c by Cr2+. In the original reports, the Cr3+ which is formed following reductioi of the protein was found to form a stable substitution-inert complex with the protein (54, 55) that appeared to involve a crosslink between Asn-52 and Tyr-67 (55). From this ohservation, it was concluded that these residues are involved in the Cr2+reduction pathway. This location of Cr3+ binding to cytochrome c has since been questioned by NMR studies, however (56). Pecht and Farver have studied the Cr3+-protein product formed on reduction of plastocyanin and azurin by Cr2+(51.53) With plasturyanin, CrJ' forms a stable 1:l complex that pept~demnppine results indicated orobablv involved two carboxylate iigands from residues 40-49 (52j. This site places the Cr3+adjacent to Tyr-83, approximately 12-15 A from the copper center. Changes in the fluorescence emission spectrum of plastocyanin and its variation with pH produced by Cr3+ binding (azurin contains no tryptophan) further implicated a site adjacelrt to a tyrosine residue (52). has been emnloved A variant of this annroach .. . , (57) . . bv . the use ufCr '-modified plasruryanin ~uevaluutctheextent of overlap brtwrrn the Cr" hindine- site and that of other inoreanir reagents. Considerable progress has been made in the analvsis of hinding interact iunghetween redox-active proteins inirrent years. One mnjur stimulus to this work has been acomputergraphics-generated model proposed by Salemme for ahypothetical complex formed between cytochrome c and cytochrome b~ (58).This model was developed by examination of the three-dimensional structures of the two (oppositely charged) proteins and optimization of the electrostatic interaction between the surfaces of the proteins surrounding the partially exposed heme edge. The resulting complex involved four electrostatic interactions between carboxyl groups on cytochrome b~ and amino arouos on cvtochrome c. As aconsequence of this optimization of'the ch;rge interaction between the proteins, the heme moups of the two cvtochromes are nearly coplanar and their-surface-accessible edges are approximately 7.5 A apart. Subsequent gel filtration and ultracentrifugation studies by Millett and co-workers substantiated the existence of this complex in solution (11) while optical difference spectroscopy was used by Mauk, et al. (59), to determine the stability of complex formation under conditions of varying pH, temperature, and ionic strength. A similar com~uter-graphics model for the comolex formed between c;tochr&ne c and cytochrome c peroxidase has been proposed by Poulos and Kraut (60). On intriguing feature of this model is the suggestion that a Phe residue (87 in yeast cytochrome c) and a His residue (181 in cvtochrome c peroxidase) are positioned between the heme (heme
a
~
~~~~~~~~~~
-
~~~~
Figure 3. Computer-generated graphical representation of the proposed structure of the cytochrome bs-hemoglobin complex. This is the complex proposed to be involved In the methemaglabin reductase activity of cytochrome bs (see ref [64]).
edge-to-heme edge distances -18 A) and assist electron transfer between the two proteins. With the availability of the refined structure of cytochrome c oeroxidase. a revised model for the comnlex has recently hken proposed in which the direct involv&nent of Phe-82 in electron transfer is eliminated (61) (vide infra). Association constants for the stability of this complex have not been reported, but chemical evidence has been nrovided to support Home of the structural features of this model (62). More recently, the stability of the interaction between cytochrome bs and methemogiobin has been studied at equilibrium (63), and a computer-graphics model for this complex is shown in Figure 3 (64). Although some details of the three computer-graphics generated models developed so far are unique to the individual complexes, the similarities between them are striking. Of particular significance is the (near) coolanaritv of the heme erouos . in the interactine proteins, the relative1 short heme edge-to-heme edge distances involved (7-18 ),and the importance of electrostatic contacts between proteins. Although much remains to be learned about the effects of protein-protein interaction on the mechanisms of electron transfer reactions between metalloproteins, the information that is available has been of considerable use in the design of experiments to study electron transfer between metalloproteins within preformed complexes. The strategy in this work has been to replace the iron heme in one of the two interactine rotei ins with Zn-orotooorohvrin . . . IX and then use nuked laser irradiation to iiduce electron transfer from the bhotoinduced 3Zn-nor~hvrincenter. which will have a verv low reduction poien
