Cytochrome C-551i Research Articles

Copper-binding energetics of amicyanin in different folding states.

Amicyanin is a type I copper protein that mediates electron transfer between methylamine dehydrogenase and cytochrome c-551i for energy production in Paracoccus denitrificans. Although the Met98 axial ligand of amicyanin has been shown to dictate metal selectivity and specificity during protein folding, the mechanism involved in copper-mediated amicyanin folding is unknown. Here, we kinetically and spectroscopically described reaction steps for incorporating copper into fully and less folded apo-amicyanin and established thermodynamic parameters for two amicyanin folding states. The rate constant for the incorporation of copper into fully folded apo-amicyanin at 25 °C was almost 1.5-fold lower than that for the initial phase of copper addition to the less folded apo-amicyanin. However, the rate constant was 10-fold higher than that of the second phase of copper addition to less folded apo-amicyanin at 25 °C. When overall binding energetic parameters (ΔH° and ΔS°) for the incorporation of copper into fully folded apo-amicyanin were measured by the van't Hoff method and isothermal titration calorimetry, the values were more positive than those determined for less folded apo-amicyanin. This indicates that during amicyanin biogenesis, copper rapidly binds to an unfolded apo-amicyanin active site, inducing protein folding and favorably influencing subsequent organization of copper ligands.

Intra-electron transfer of amicyanin from newly derived active site to redox potential tuned type 1 copper site

Amicyanin, one of the type I copper proteins which has been used for the study, mediates the electron transfer reaction between methylamine dehydrogenase and cytochrome c-551i in Paracoccus denitrificans for energy production. The 6×Histidine-tag site which has been widely used in purification of a recombinant protein was introduced at the N-terminus of amicyanin to make the complex of 6×His-tagged plus cobalt functioning as a newly derived redox cofactor in amicyanin. In this study, Pro94 of amicyanin was substituted to Ala and Phe to tune up the midpoint potential (Em) value of amicyanin 100 mV more positive and then intra-electron transfer rates were measured to examine whether the Em value of the type 1 copper site in amicyanin affects intraprotein electron transfer or not. By the addition of H2O2, the Co2+-loaded 6×His-tagged site was activated, and then electron was transferred from Cu1+ of type 1 copper site of amicyanin to Co3+ plus 6×His-tagged site. Electron transfer rates of cobalt loaded P94A and F amicyanin were much slower than that of native amicyanin. These results suggest that the communication between the newly protein-derived redox cofactor, 6×His-tagged site plus cobalt, and type 1 copper site is truly occurred and that the strength of electron transfer reaction between them is able to be controlled by an Em value.

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.

Amicyanin Transfers Electrons from Methylamine Dehydrogenase to Cytochrome c-551i via a Ping-Pong Mechanism, not a Ternary Complex

The first crystal structure of a ternary redox protein complex was comprised of the enzyme methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans [Chen et al. Science 1994, 264, 86-90]. The arrangement of the proteins suggested possible electron transfer from the active site of MADH via the amicyanin copper ion to the cytochrome heme iron, although the distance between the metals is large. We studied the interactions between these proteins in solution. A titration followed by NMR spectroscopy shows that amicyanin binds cytochrome c-551i. The interface comprises the hydrophobic and positive patches of amicyanin, not the binding site observed in the ternary complex. NMR experiments further show that amicyanin binds tightly to MADH with an interface that matches the one observed in the crystal structure and that mostly overlaps with the binding site for cytochrome c-551i. Upon addition of cytochrome c-551i, no changes in the NMR spectrum of MADH-bound amicyanin are observed, suggesting that a possible interaction of the cytochrome with the binary complex must be very weak, with a dissociation constant higher than 2 mM. Reconstitution of the entire redox chain in vitro demonstrates that amicyanin can react rapidly with cytochrome c-551i, but that association of amicyanin with MADH inhibits this reaction. It is concluded that electron transfer from MADH to cytochrome c-551i does not involve a ternary complex but occurs via a ping-pong mechanism in which amicyanin uses the same interface for the reactions with MADH and cytochrome c-551i.

ChemInform Abstract: What Controls the Rates of Interprotein Electron-Transfer Reactions?

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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.

Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome c551i as detected by EPR spectroscopy.

EPR studies of the methylamine dehydrogenase (MADH)-amicyanin and MADH-amicyanin-cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry. The binary complex shows a blue copper axial signal, characteristic of oxidized amicyanin. After reaction of substrate with the MADH coenzyme tryptophan tryptophylquinone (TTQ), the binary complex exhibits an equilibrium mixture of oxidized copper/reduced TTQ and reduced copper/TTQ. radical, whose ratio is dependent on the pH. In the oxidized ternary complex, the same copper axial signal is observed superimposed on the low-spin ferric heme features characteristic of oxidized cytochrome c551i. After addition of substrate to the ternary complex, a decrease of the copper signal is observed, concomitant with the appearance of the radical signal derived from the semiquinone form of TTQ. The equilibrium distribution of electrons between TTQ and copper as a function of pH is similar to that observed for the binary complex. This result was essential to establish that the copper center retains its function within the crystalline ternary complex. At high pH, with time the low-spin heme EPR features disappear and the spectrum indicates that full reduction of the complex by substrate has occurred.

X-ray structure of methanol dehydrogenase from Paracoccus denitrificans and molecular modeling of its interactions with cytochrome c-551i

The X-ray structure of methanol dehydrogenase (MEDH) from Paracoccus denitrificans (MEDH-PD) was determined at 2.5 A resolution using molecular replacement based on the structure of MEDH from Methylophilus methylotrophus W3A1 (MEDH-WA). The overall structures from the two bacteria are similar to each other except that the former has a longer C-terminal tail in each subunit and shows local differences in several insertion regions. The "X-ray sequence" of the segment alphaGly444-alphaLeu452 was established, including one insertion and seven replacements compared with the reported sequence. The primary electron acceptor of MEDH-PD is cytochrome c-551i (Cyt c551i). Based on the crystal structure of MEDH-PD and of the published structure of Cyt c551i, their interactions were investigated by molecular modeling. As a guide and starting point, the covalently attached cytochrome and PQQ domains of the alcohol dehydrogenase from Pseudomonas putida HK5 (ADH2B) were used. In the modeling, two molecules of Cyt c551i could be accommodated in their interaction with the MEDH heterotetramer in accordance with the two-fold molecular symmetry of the latter. Two models are proposed, in both of which electrostatic and hydrogen bonding interactions make major contributions to inter-protein binding. One of these models involves salt bridges from alphaArg99 of MEDH to the heme propionic acids of Cyt c551i and the other involves salt bridges from alphaArg426 of MEDH to Glu112 of Cyt c551i. Both involve salt bridges from alphaLys93 of MEDH to Asp75 of Cyt c551i. The size and nature of the cytochrome/quinoprotein heterodimer interfaces and calculations of electronic coupling and electron transfer rates favor one of these models over the other.

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan–tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H AB, related to electronic coupling between the two centers, and λ, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter β, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.

Effects of engineering uphill electron transfer into the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex.

Within the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex, electrons are transferred from tryptophan tryptophylquinone (TTQ) to heme via the type I copper center of amicyanin. Mutation of Pro94 of amicyanin to Phe increases the redox potential of the copper center within the protein complex by approximately 195 mV. This introduces a large energy barrier for the second electron transfer (ET) step in this three-protein ET chain. As a consequence of this mutation, the ET rate from TTQ to copper exhibits about a 6-fold increase and the ET rate from copper to heme exhibits about a 100-fold decrease. These changes in ET rate are consistent with the predictions of Marcus theory. Temperature dependence studies of these reactions indicate that the reorganization energies for the ET to and from the copper center are unchanged by the P94F mutation, despite the large change in redox potential that it causes. Steady-state kinetic studies indicate that despite the large energy barrier for the ET from copper to heme, methylamine-dependent reduction of heme by the three-protein complex with P94F amicyanin goes to completion. The turnover number for this steady-state reaction, however, is decreased 50-fold relative to that of the native complex. As a consequence of the P94F mutation, the rate constant for the unfavorable uphill ET reaction from copper to heme has become the rate-limiting step in the overall reaction. The evolutionary implications of the effects of this mutation on the function of this naturally occurring simple ET chain are discussed.

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.

Tyr 30 of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways

A Pathways analysis of the methylamine dehydrogenase–amicyanin–cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cys 92 copper ligand, and in the other, it exits via the Met 98 copper ligand. If the Pathways algorithm is modified to include contributions from the anisotropy of metal–ligand coupling, independent of differences in copper–ligand bond length, then the pathways via Cys 92 are predicted to be at least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys 92 include a through-space jump from Cys 92 to the side chain of Tyr 30. To determine whether or not the pathways via Cys 92 are preferentially used for ET, Tyr 30 was changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr 30 in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors ET via Cys 92 predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met 98 is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.

Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin.

The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.

Enzymatic and Electron Transfer Activities in Crystalline Protein Complexes

Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme.

Electron Transfer from Copper to Heme within the Methylamine Dehydrogenase−Amicyanin−Cytochrome c-551i Complex

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution [Chen, L., Durley, R., Mathews, F. S., & Davidson, V. L. (1994) Science 264, 86-90] which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The electron transfer reaction from copper to heme within the protein complex has been characterized by transient kinetic and thermodynamic analysis. The rate of this electron transfer reaction is 87 s-1 at 30 degrees C, and it varied with temperature. The reaction exhibited a reorganizational energy (lambda) of 1.1 eV and an electronic coupling (H(AB)) of 0.3 cm-1. The results of these analyses also predict an electron transfer distance, depending upon the value of beta which is used, of 13-24 A. The larger value approximates the direct copper to heme distance observed in the crystal structure of the complex. The most efficient pathways for electron transfer were predicted from the crystal structure using the Greenpath program, and these predictions were correlated with the results of the solution studies of the electron transfer reaction. It is concluded that electron transfer is, in fact, rate limiting for the observed electron transfer reaction in solution and that the two redox centers are strongly coupled, given the distance which separates them.

Complex Formation with Methylamine Dehydrogenase Affects the Pathway of Electron Transfer from Amicyanin to Cytochrome c-551i

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution (Chen, L., Durley, R., Mathews, F. S., and Davidson, V. L. (1994) Science 264, 86-90), which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The reduction of cytochrome by amicyanin within the complex in solution has been demonstrated using rapid scanning stopped-flow spectroscopy. Electron transfer from free, uncomplexed, amicyanin to cytochrome c-551i occurs much more rapidly but only to a very small extent because the reaction is thermodynamically much less favorable when amicyanin is not associated with MADH (Gray, K. A., Davidson, V. L., and Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990). These kinetic data suggest that amicyanin binding to cytochrome c-551i occurs at different sites when amicyanin is free and when it is in complex with MADH. A model for the interactions of these proteins is presented.

Roles of dipolar effects and local charge in the ionic strength dependence of redox reactions between c-type cytochromes

Redox reactions between different c-type cytochromes were monitored by stopped-flow spectroscopy. Second-order rate constants were determined at different ionic strengths for the reactions of Paracoccus denitrificans cytochrome c-551i with its physiologic redox partner cytochrome c-550, and with the nonphysiologic partner horse heart cytochrome c. The latter two cytochromes are structurally quite similar and exhibit identical redox potentials but bear net charges of -7 and +7, respectively. Despite these opposite overall charges, the ionic strength dependencies for the reaction of each with the acidic cytochrome c-551i were very similar. The observed decrease in reaction rate with increasing ionic strength that was observed with cytochromes c-550 and c-551i, the latter of which bears a net charge of -20, cannot be explained simply on the basis of monopole-monopole interactions. These data were analyzed by two different methods: one which treats proteins as both monopoles and dipoles and considers the net charge; and another which neglects dipolar effects and considers only the local charge of the reactive site of the protein rather than net charge. The applicability of each method to the analysis of these data and to protein electrostatic interactions in general is discussed.

Ionic strength dependence of the reaction between methanol dehydrogenase and cytochrome c-551i: evidence of conformationally coupled electron transfer.

The quinoprotein methanol dehydrogenase and cytochrome c-551i are two soluble acidic proteins that form a physiological complex in which electrons are transferred from pyrroloquinoline quinone to heme. The oxidation of methanol dehydrogenase by the cytochrome was studied as a function of ionic strength using stopped-flow spectroscopy. The dissociation constant (Kd) for complex formation decreased 2-fold with increasing ionic strength from 0.21 to 1.3 M and increased at higher ionic strengths. The rate constant for the electron transfer reaction (kET) increased 2-fold with increasing ionic strength from 0.21 to 1.3 M and decreased at higher ionic strengths. The variation of Kd and kET over this range of ionic strengths was described by Van Leeuwen theory, which takes into account monopole-dipole and dipole-dipole forces, in addition to the monopole-monopole force, to predict the interactions between large molecules. Analysis of the kinetic results in terms of these electrostatic interactions indicated the probable orientations for protein-protein binding and electron transfer. To explain the ionic strength dependence of the observed kET, a model is presented in which the true kET is reduced by a factor Kc, an equilibrium constant that describes some rearrangement of the proteins after a nonoptimal collision to produce the most efficient orientation for electron transfer. This model is consistent with the notion that the large reorganizational energy obtained from temperature-dependence studies of this electron transfer reaction [Harris, T. K., & Davidson, V. L. (1993) Biochemistry 32, 14145-14150] is due to such an intracomplex rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of an electron transfer complex: methylamine dehydrogenase, amicyanin, and cytochrome c551i.

The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c551i. The central region of the c551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.

Binding and electron transfer reactions between methanol dehydrogenase and its physiologic electron acceptor cytochrome c-551i: a kinetic and thermodynamic analysis.

The quinoprotein methanol dehydrogenase and cytochrome c-551i form a physiologic complex in which electrons are transferred from pyrroloquinoline quinone to heme. The reoxidation of methanol dehydrogenase by the cytochrome was studied by stopped-flow spectroscopy. The rate constant for the electron transfer reaction and the dissociation constant for complex formation were each determined at temperatures ranging from 20 to 50 degrees C. The electron transfer rates varied from 1.4 to 4.6 s-1. Analysis of the electron transfer reaction by Marcus theory yielded values of 1.9 eV for the reorganizational energy and 0.071 cm-1 for the electronic coupling and predicted a theoretical distance between redox centers of 15 A. Kinetically determined dissociation constants correlated well with a Kd of 375 microM which was determined in a direct ultrafiltration binding assay. Thermodynamic analysis of the dissociation constants indicated the importance of the hydrophobic effect in complex formation.