True Electron Transfer Research Articles

Charge-Disproportionation Symmetry Breaking Creates a Heterodimeric Myoglobin Complex with Enhanced Affinity and Rapid Intracomplex Electron Transfer.

We report rapid photoinitiated intracomplex electron transfer (ET) within a "charge-disproportionated" myoglobin (Mb) dimer with greatly enhanced affinity. Two mutually supportive Brownian Dynamics (BD) interface redesign strategies, one a new "heme-filtering" approach, were employed to "break the symmetry" of a Mb homodimer by pairing Mb constructs with complementary highly positive and highly negative net surface charges, introduced through D/E → K and K → E mutations, respectively. BD simulations using a previously developed positive mutant, Mb(+6) = Mb(D44K/D60K/E85K), led to construction of the complementary negative mutant Mb(-6) = Mb(K45E, K63E, K95E). Simulations predict the pair will form a well-defined complex comprising a tight ensemble of conformations with nearly parallel hemes, at a metal-metal distance ∼18-19 Å. Upon expression and X-ray characterization of the partners, BD predictions were verified through ET photocycle measurements enabled by Zn-deuteroporphyrin substitution, forming the [ZnMb(-6), Fe(3+)Mb(+6)] complex. Triplet ET quenching shows charge disproportionation increases the binding constant by no less than ∼5 orders of magnitude relative to wild-type Mb values. All progress curves for charge separation (CS) and charge recombination (CR) are reproduced by a generalized kinetic model for the interprotein ET photocycle. The intracomplex ET rate constants for both CS and CR are increased by over 5 orders of magnitude, and their viscosity independence is indicative of true interprotein ET, rather than dynamic gating as seen in previous studies. The complex displays an unprecedented timecourse for CR of the CS intermediate I. After a laser flash, I forms through photoinduced CS, accumulates to a maximum concentration, then dies away through CR. However, before completely disappearing, I reappears without another flash and reaches a second maximum before disappearing completely.

Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis

The interprotein electron transfer (ET) reactions of the cupredoxin amicyanin, which mediates ET from the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase to cytochrome c-551i have been extensively studied. However, it was not possible to perform certain key experiments in that native system. This study examines the ET reaction from reduced amicyanin to an alternative electron acceptor, the diheme protein MauG. It was possible to vary the ΔG° for this ET reaction by simply changing pH to determine the dependence of kET on ΔG°. A P94A mutation of amicyanin significantly altered its oxidation–reduction midpoint potential value. It was not possible to study the ET from reduced P94A amicyanin to cytochrome c-551i in the native system because that reaction was kinetically coupled. However, the reaction from reduced P94A amicyanin to MauG was a true ET reaction and it was possible to determine values of reorganization energy (λ) and electronic coupling for the reactions of this variant as well as native amicyanin. Comparison of the λ values associated with the ET reactions between amicyanin and the TTQ of methylamine dehydrogenase, the diheme center of MauG and the single heme of cytochrome c-551i, provides insight into the factors that dictate the λ values for the respective reactions. These results demonstrate how study of ET reactions with alternative redox partner proteins can complement and enhance our understanding of the reactions with the natural redox partners, and further our understanding of mechanisms of protein ET reactions.

Photocharging Artifacts in Measurements of Electron Transfer in Quantum-Dot-Sensitized Mesoporous Titania Films.

Transient absorption and time-resolved photoluminescence measurements of high-performance mesoporous TiO2 photoanodes sensitized with CuInSexS2-x quantum dots reveal the importance of hole scavenging in the characterization of photoinduced electron transfer. The apparent characteristic time of this process strongly depends on the local environment of the quantum dot/TiO2 junction due to accumulation of long-lived positive charges in the quantum dots. The presence of long-lived photoexcited holes introduces artifacts due to fast positive-trion Auger decay (60 ps time constant), which can dominate electron dynamics and thus mask true electron transfer. We show that the presence of a redox electrolyte is critical to the accurate characterization of charge transfer, since it enables fast extraction of holes and helps maintain charge neutrality of the quantum dots. Although electron transfer is observed to be relatively slow (19 ns time constant), a high electron extraction efficiency (>95%) can be achieved because in well-passivated CuInSexS2-x quantum dots neutral excitons have significantly longer lifetimes of hundreds of nanoseconds.

Proline 96 of the Copper Ligand Loop of Amicyanin Regulates Electron Transfer from Methylamine Dehydrogenase by Positioning Other Residues at the Protein−Protein Interface

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a "hydrophobic patch" of amino acid residues including those that comprise a "ligand loop" that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K(d) for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H(AB)) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin, but notably, in addition to the change in Pro96, the side chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K(d), λ, and H(AB) for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner but also to optimize the orientation of proteins for interprotein ET.

Molecular Basis for Directional Electron Transfer

Biological macromolecules involved in electron transfer reactions display chains of closely packed redox cofactors when long distances must be bridged. This is a consequence of the need to maintain a rate of transfer compatible with metabolic activity in the framework of the exponential decay of electron tunneling with distance. In this work intermolecular electron transfer was studied in kinetic experiments performed with the small tetraheme cytochrome from Shewanella oneidensis MR-1 and from Shewanella frigidimarina NCIMB400 using non-physiological redox partners. This choice allowed the effect of specific recognition and docking to be eliminated from the measured rates. The results were analyzed with a kinetic model that uses the extensive thermodynamic characterization of these proteins reported in the literature to discriminate the kinetic contribution of each heme to the overall rate of electron transfer. This analysis shows that, in this redox chain that spans 23 A, the kinetic properties of the individual hemes establish a functional specificity for each redox center. This functional specificity combined with the thermodynamic properties of these soluble proteins ensures directional electron flow within the cytochrome even outside of the context of a functioning respiratory chain.

Protein control of true, gated, and coupled electron transfer reactions.

Electron transfer (ET) through and between proteins is a fundamental biological process. The rates of ET depend upon the thermodynamic driving force, the reorganization energy, and the degree of electronic coupling between the reactant and product states. The analysis of protein ET reactions is complicated by the fact that non-ET processes might influence the observed ET rate in kinetically complex biological systems. This Account describes studies of the methylamine dehydrogenase-amicyanin-cytochrome c-551i protein ET complex that have revealed the influence of several features of the protein structure on the magnitudes of the physical parameters for true ET reactions and how they dictate the kinetic mechanisms of non-ET processes that sometimes influence protein ET reactions. Kinetic and thermodynamic studies, coupled with structural information and biochemical data, are necessary to fully describe the ET reactions of proteins. Site-directed mutagenesis can be used to elucidate specific structure-function relationships. When mutations selectively alter the electronic coupling, reorganization energy, or driving force for the ET reaction, it becomes possible to use the parameters of the ET process to determine how specific amino acid residues and other features of the protein structure influence the ET rates. When mutations alter the kinetic mechanism for ET, one can determine the mechanisms by which non-ET processes, such as protein conformational changes or proton transfers, control the rates of ET reactions and how specific amino acid residues and certain features of the protein structure influence these non-ET reactions. A complete description of the mechanism of regulation of biological ET reactions enhances our understanding of metabolism, respiration, and photosynthesis at the molecular level. Such information has important medical relevance. Defective protein ET leads to production of the reactive oxygen species and free radicals that are associated with aging and many disease states. Defective ET within the respiratory chain also causes certain mitochondrial myopathies. An understanding of the mechanisms of regulation of protein ET is also of practical value because it provides a logical basis for the design of applications utilizing redox enzymes, such as enzyme-based electrode sensors and fuel cells.

A Single Methionine Residue Dictates the Kinetic Mechanism of Interprotein Electron Transfer from Methylamine Dehydrogenase to Amicyanin,

Amicyanin is a type 1 copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). A P52G amicyanin mutation increased the Kd for complex formation and caused the normally true electron transfer (ET) reaction from O-quinol MADH to amicyanin to become a gated ET reaction (Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284-8293). One consequence of the P52G mutation was to reposition the side chain of Met51, which is present at the MADH-amicyanin interface. To examine the precise role of Met51 in this interprotein ET reaction, Met51 was converted to Ala, Lys, and Leu. The Kd for complex formation of M51A amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm-1 to 142 cm-1, and the reorganization energy increased from 2.3 to 3.1 eV. The rate and salt dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin is also changed by the M51A mutation. These changes in ET parameters and rates for the reactions with M51A amicyanin were similar to those caused by the P52G mutation and indicated that the ET reaction had become gated by a similar process, most likely a conformational rearrangement of the protein ET complex. The results of the M51K and M51L mutations also have consequences on the kinetic mechanism of regulation of the interprotein ET with effects that are intermediate between what is observed for the reaction of the native amicyanin and M51A amicyanin. These data indicate that the loss of the interactions involving Pro52 were primarily responsible for the change in Kd for P52G amicyanin, while the interactions involving the Met51 side chain are entirely responsible for the change in ET parameters and conversion of the true ET reaction of native amicyanin into a conformationally gated ET reaction.

Correlation of Rhombic Distortion of the Type 1 Copper Site of M98Q Amicyanin with Increased Electron Transfer Reorganization Energy

Mutation of the axial Met ligand of the type 1 copper site of amicyanin to Ala or Gln yielded M98A amicyanin, which exhibits typical axial type 1 ligation geometry but with a water molecule providing the axial ligand, and M98Q amicyanin, which exhibits significant rhombic distortion of the type 1 site (Carrell, C. J., Ma, J. K., Antholine, W. E., Hosler, J. P., Mathews, F. S., and Davidson, V. L. (2007) Biochemistry 46, 1900-1912). Despite the change of the axial ligand, the M98Q and M98A mutations had little effect on the redox potential of copper. The true electron transfer (ET) reactions from O-quinol methylamine dehydrogenase to oxidized native and mutant amicyanins revealed that the M98A mutation had little effect on kET, but the M98Q mutation reduced kET 45-fold. Thermodynamic analysis of the latter showed that the decrease in kET was due to an increase of 0.4 eV in the reorganization energy (lambda) associated with the ET reaction to M98Q amicyanin. No change in the experimentally determined electronic coupling or ET distance was observed, confirming that the mutation had not altered the rate-determining step for ET and that this was still a true ET reaction. The basis for the increased lambda is not the nature of the atom that provides the axial ligand because each uses an oxygen from Gln in M98Q amicyanin and from water in M98A amicyanin. Comparisons of the distance of the axial copper ligand from the equatorial plane that is formed by the other three copper ligands in isomorphous crystals of native and mutant amicyanins at atomic resolution indicate an increase in distance from 0.20 A in the native to 0.42 A in M98Q amicyanin and a slight decrease in distance for M98A amicyanin. This correlates with the rhombic distortion caused by the M98Q mutation that is clearly evident in the EPR and visible absorption spectra of the protein and suggests that the extent of rhombicity of the type 1 copper site influences the magnitude of lambda.

Site-Directed Mutagenesis of Proline 52 To Glycine in Amicyanin Converts a True Electron Transfer Reaction into One that Is Conformationally Gated,

Amicyanin is a type I copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). The conversion of Proline52 of amicyanin to a glycine does not alter the physical and spectroscopic properties of the copper binding site, but it does alter the rate of electron transfer (ET) from MADH. The values of electronic coupling (H(AB)) and reorganization energy (lambda) that are associated with the true ET reaction from the reduced O-quinol tryptophan tryptophylquinone (TTQ) of MADH to oxidized amicyanin are significantly altered as a consequence of the P52G mutation. The experimentally determined H(AB) increases from 12 to 78 cm(-1), and lambda increases from 2.3 to 2.8 eV. The rate and salt-dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin are also changed by the P52G mutation. Kinetic data suggests that a new common reaction step has become rate-limiting for both the true and gated ET reactions that occur from different redox forms of MADH. A comparison of the crystal structures of P52G amicyanin with those of native amicyanin free and in complex with MADH provided clues as to the basis for the change in ET parameters. The mutation results in the loss of three carbons from Pro52 and the movement of the neighboring residue Met51. This reduces the number of hydrophobic interactions with MADH in the complex and perturbs the protein-protein interface. A model is proposed for the ET reaction with P52G amicyanin in which the most stable conformation of the protein-protein complex with MADH is not optimal for ET. A new preceding kinetic step is introduced prior to true ET that requires P52G amicyanin to switch from this redox-inactive stable complex to a redox-active unstable complex. Thus, the ET reaction of P52G amicyanin is no longer a true ET but one that is conformationally gated by the reorientation of the proteins within the ET protein complex. This same reaction step now also gates the ET from N-quinol MADH, which is normally rate-limited by a proton transfer.

Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum. Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

The thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of alpha-hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 A, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s(-1) and is kinetically gated by the preceding formation of the tetrahedral substrate-ThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s(-1) and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FAD(ox)/FAD(sq) (E1 = -37 mV) and FAD(sq)/FAD(red) (E2 = -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3'-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 10(2)-10(3)-fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosphorolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.

Site-Directed Mutagenesis of Proline 94 to Alanine in Amicyanin Converts a True Electron Transfer Reaction into One That Is Kinetically Coupled

Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro(94) resides in the "ligand loop" of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (H(AB)) and reorganization energy (lambda) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of H(AB) and lambda are 8.3 cm(-)(1) and 2.3 eV, respectively, compared to values of 0.3 cm(-)(1) and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry 43, 9372-9380]. In one of these, conformation B, a water molecule has replaced Met(98) as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 A. Analysis of these structures suggests that the true k(ET) for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu(2+) by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu(1+) by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the K(eq) and values of DeltaH degrees and DeltaS degrees that are associated with the non-ET reaction that modulates the observed ET rate.

Asymmetric salt effects on anion/cation reactions: A comparative study of the [Fe(CN)6]4− + [Co(NH3)5pz]3+ and [Ru(NH3)5pz]2+ + [Co(C2O4)3]3− reactions

AbstractThe kinetics of electron transfer reactions between [Fe(CN)6]4− and [Co(NH3)5pz]3+ and between [Ru(NH3)5pz]2+ and [Co(C2O4)3]3− was studied in concentrated salt solutions (Na2SO4, LiNO3, and Ca(NO3)2). An analysis of the experimental kinetic data, kobs, permits us to obtain the true (unimolecular) electron transfer rate constants corresponding to the true electron transfer process (precursor complex → successor complex), ket. The variations of both, kobs and ket, with salt concentrations are opposite for these reactions. These opposite tendencies can be rationalized by using the Marcus–Hush treatment for electron transfer reactions. The conclusion is that the negative salt effect found for the first reaction ([Fe(CN)6]4− + [Co(NH3)5pz]3+) is due to the increase of the reaction and reorganization free energies when the concentration of salt increases. In the case of the second reaction ([Ru(NH3)5pz]2+ + [Co(C2O4)3]3−), the positive salt effect observed is caused by the fact that the driving force becomes more favorable when the concentration of salt increases. Thus, it is shown that for anion/cation electron transfer reactions the kinetic salt effect depends on the charge sign of the oxidant (and the reductant). © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 37: 81–89, 2005

Metalloprotein association, self-association, and dynamics governed by hydrophobic interactions: simultaneous occurrence of gated and true electron-transfer reactions between cytochrome f and cytochrome c(6) from Chlamydomonas reinhardtii.

Noninvasive reconstitution of the heme in cytochrome c(6) with zinc(II) ions allowed us to study the photoinduced electron-transfer reaction (3)Zncyt c(6) + cyt f(III) --> Zncyt c(6)(+) + cyt f(II) between physiological partners cytochrome c(6) and cytochrome f, both from Chlamydomonas reinhardtii. The reaction kinetics was analyzed in terms of protein docking and electron transfer. In contrast to various protein pairs studied before, both the unimolecular and the bimolecular reactions of this oxidative quenching take place at all ionic strengths from 2.5 through 700 mM. The respective intracomplex rate constants are k(uni) (1.2 +/- 0.1) x 10(4) s(-1) for persistent and k(bi) (9 +/- 4) x 10(2) s(-1) for the transient protein complex. The former reaction seems to be true electron transfer, and the latter seems to be electron transfer gated by a structural rearrangement. Remarkably, these reactions occur simultaneously, and both rate constants are invariant with ionic strength. The association constant K(a) for zinc cytochrome c(6) and cytochrome f(III) remains (5 +/- 3) x 10(5) M(-1) in the ionic strength range from 700 to 10 mM and then rises slightly to (7 +/- 2) x 10(6) M(-1), as ionic strength is lowered to 2.5 mM. Evidently, docking of these proteins from C. reinhardtii is due to hydrophobic interaction, slightly augmented by weak electrostatic attraction. Kinetics, chromatography, and cross-linking consistently show that cytochrome f self-dimerizes at ionic strengths of 200 mM and higher. Cytochrome f(III) quenches triplet state (3)Zncyt c(6), but its dimer does not. Formation of this unreactive dimer is an important step in the mechanism of electron transfer. Not only association between the reacting proteins, but also their self-association, should be considered when analyzing reaction mechanisms.

The heme domain of cellobiose oxidoreductase: a one-electron reducing system

Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases.

Evidence for substrate activation of electron transfer from methylamine dehydrogenase to amicyanin.

Electron transfer (ET) from methylamine dehydrogenase (MADH) to amicyanin may be true or gated ET, depending upon the redox form of MADH. ET from the substrate-reduced aminoquinol form of MADH is gated, and the reaction rate is dependent on the presence of monovalent cations. This ET reaction has been studied in buffer free of monovalent cations. The reaction rate is orders of magnitude less than with saturating concentrations of monovalent cation. Analysis of the temperature dependence of this slow reaction, however, reveals that it is a true ET reaction. The rate of MADH reduction by substrate and the steady-state rate of substrate-dependent reduction of amicyanin by MADH were examined in different buffers. The results reveal that, in the steady state, the protonated methylammonium substrate performs the role previously attributed to monovalent cations in regulating the rate and mechanism of ET from MADH. The two putative cation binding sites previously observed in the crystal structure of MADH may now be assigned distinct roles, one as a catalytic substrate binding site and the other as a noncatalytic regulatory substrate binding site.

油水界面電子移動の反応機構に関する研究

The study of electron transfer (ET) at the oil (O)/water (W) interface is useful for understanding energy conversion processes occurring at biomembranes. The recent introduction of SECM (scanning electrochemical microscopy) as well as other new methods to this field has revitalized the study of the reaction mechanism of ET at the O/W interface. However, the mechanism is not necessarily simple, and often involves an ion-transfer process. When a not very hydrophobic complex such as ferrocene (Fc) is used for the redox species in the O phase, the ET would occur homogeneously in the W phase, the interfacial transfer of the ionic product (e.g., Fc+) being responsible for the current flowing through the interface. On the other hand, when an extremely hydrophobic complex such as lutetium bihthalocyanine is used, the heterogeneous, i.e., true ET should occur at the O/W interface. Marcus proposed a general kinetic equation for the heterogeneous ET. There are some experimental data which support the Marcus theory, however, further careful and detailed studies seem to be required. In this review, recent developments in theory and methodology in this research field are described.

Mutation of alphaPhe55 of methylamine dehydrogenase alters the reorganization energy and electronic coupling for its electron transfer reaction with amicyanin.

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis of alphaPhe55 has revealed roles for this residue in determining substrate specificity and binding monovalent cations at the active site. It is now shown that the alphaF55A mutation also increases the rate of the true electron transfer (ET) reaction from O-quinol MADH to amicyanin. The reorganization energy associated with the ET reaction is decreased from 2.3 to 1.8 eV. The electronic coupling associated with the ET reaction is decreased from 12 to 3 cm(-1). The crystal structure of alphaF55A MADH in complex with its electron acceptors, amicyanin and cytochrome c-551i, has been determined. Little difference in the overall structure is seen, relative to the native complex; however, there are significant changes in the solvent content of the active site and substrate channel. The crystal structure of alphaF55A MADH has also been determined with phenylhydrazine covalently bound to TTQ in the active site. Phenylhydrazine binding significantly perturbs the orientation of the TTQ rings relative to each other. The ET results are discussed in the context of the new and old crystal structures of the native and mutant enzymes.

Photoinduced Electron Transfer between the Rieske Iron-Sulfur Protein and Cytochrome c1 in theRhodobacter sphaeroides Cytochromebc1 Complex: EFFECTS OF pH, TEMPERATURE, AND DRIVING FORCE

Electron transfer from the Rieske iron-sulfur protein to cytochrome c(1) (cyt c(1)) in the Rhodobacter sphaeroides cytochrome bc(1) complex was studied using a ruthenium dimer complex, Ru(2)D. Laser flash photolysis of a solution containing reduced cyt bc(1), Ru(2)D, and a sacrificial electron acceptor results in oxidation of cyt c(1) within 1 micros, followed by electron transfer from the iron-sulfur center (2Fe-2S) to cyt c(1) with a rate constant of 80,000 s(-1). Experiments were carried out to evaluate whether the reaction was rate-limited by true electron transfer, proton gating, or conformational gating. The temperature dependence of the reaction yielded an enthalpy of activation of +17.6 kJ/mol, which is consistent with either rate-limiting conformational gating or electron transfer. The rate constant was nearly independent of pH over the range pH 7 to 9.5 where the redox potential of 2Fe-2S decreases significantly due to deprotonation of His-161. The rate constant was also not greatly affected by the Rieske iron-sulfur protein mutations Y156W, S154A, or S154A/Y156F, which decrease the redox potential of 2Fe-2S by 62, 109, and 159 mV, respectively. It is concluded that the electron transfer reaction from 2Fe-2S to cyt c(1) is controlled by conformational gating.

Underpotential deposition of zinc ions and specific adsorption of hydroxyl species at Pt(111) in alkaline solutions

Underpotential deposition (upd) of zinc ions is observed on Pt(111) in alkaline solution as a sharp cyclic voltammogram (CV) peak, in contrast to upd on polycrystalline Pt, for which several broad upd peaks are observed. The CV characteristics on Pt(111) were studied from changes in the sweep rates, zincate ion concentrations ZnO 2 2−, and pH. The Zn upd formation/desorption mechanism is analyzed and discussed in terms of a nucleation-growth mechanism. Changes in the upd peak potential with changes in the concentration of ZnO 2 2− and OH − were 60 mV/log[ZnO 2 2−] and −180 mV/pH. The former value gives an apparent electron transfer number n a=1. The OH − adsorption/desorption peak on Pt(111) shifted with −60 mV/pH and CV peak width 90 mV, independent of sweep rate. These results show that the reaction OH −=OH ads+e − takes place according to the Langmuir adsorption mechanism. The different pH dependences for Zn upd and OH − adsorption/desorption processes suggest that the electrode reaction in alkaline solution, ZnO 2 2−+2H 2O+2e −=Zn+4OH −, is involved in upd on Pt(111). The value n a=1 for the ZnO 2 2− upd process can be discussed in terms of two-electron transfer, such that Zn 2+ is reduced to a Zn 0 state at Pt(111) with a true electron transfer number of n=2, and the induced OH − adsorption takes place as OH ads onto upd Zn at Pt(111) by an oxidative process. The overall upd process can be expressed as ZnO 2 2−+2 H 2 O+ e ⇌ Pt(111) OH|Zn|Pt+3 OH −, which is in good accord with the observed pH dependence of −180 mV/pH. Consequently, the apparent electron transfer number n a becomes 1. From the charge densities of the upd layer and the OH layer, the coverage ratio of OH ads/Zn upd is equal to 0.59/0.45 with respect to surface Pt atoms.

Diffusion, spin and reaction control in geminate reverse electron transfer

Kinetic analyses of geminate radical escape yields in terms of a simple (“exponential”) reaction scheme with first-order rate constants of separation and geminate recombination have been widely used in the literature, e.g. to evaluate rate constants of reverse electron transfer (k−et). Here we demonstrate the limited value of such rate constants by formally analysing, in terms of the exponential model, the diffusion coefficient (iz. viscosity) dependence of the radical escape yield as theoretically calculated in the framework of diffusion-dependent electron transfer theory (unified treatment of non-contact photoinduced forward and geminate reverse electron transfer). It is shown that, while the true electron transfer rate constant is kept constant, the apparent rate constant k−et from the exponential model undergoes a wide variation as a function of diffusion coefficient and the rate of spin conversion. Nevertheless, the function k−et(D) represented in a double log plot for various rates of spin conversion provides a useful map suitable to assign characteristic regions of diffusional, spin and reaction control of the geminate process. As an application to real systems the experimental example of the [Ru(bpy)3]2+/methylviologen system is reconsidered. Here a magnetic field effect on the k−et(D) dependence is useful to corroborate the non-contact formation of the radical pair in the photochemical forward electron transfer reaction.