Gated and ungated electron transfer reactions from aromatic amine dehydrogenase to azurin.

Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction by dithionite, and an N-quinol form that was generated by reduction by substrate. It was previously shown that on reduction by substrate, an amino group displaces a carbonyl oxygen on TTQ, and that this significantly alters the rate of its oxidation by azurin (Hyun, Y-L., and Davidson V. L. (1995) Biochemistry 34, 12249-12254). To determine the basis for this change in reactivity, comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper of azurin were performed. The reaction of the O-quinol exhibited values of electronic coupling (H(AB)) of 0.13 cm(-1) and reorganizational energy (lambda) of 1.6 eV, and predicted an ET distance of approximately 15 A. These results are consistent with the ET event being the rate-determining step for the redox reaction. Analysis of the reaction of the N-quinol by Marcus theory yielded an H(AB) which exceeded the nonadiabatic limit and predicted a negative ET distance. These results are diagnostic of a gated ET reaction. Solvent deuterium kinetic isotope effects of 1.5 and 3.2 were obtained, respectively, for the ET reactions from O-quinol and N-quinol AADH indicating that transfer of an exchangeable proton was involved in the rate-limiting reaction step which gates ET from the N-quinol, but not the O-quinol. These results are compared with those for the ET reactions from another TTQ enzyme, methylamine dehydrogenase, to amicyanin. The mechanism by which the ET reaction of the N-quinol is gated is also related to mechanisms of other gated interprotein ET reactions.     

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.     

Conformational reorganisation in interfacial protein electron transfer

Protein-protein electron transfer (ET) plays an essential role in all redox chains. Earlier studies which used cross-linking and increased solution viscosity indicated that the rate of many ET reactions is limited (i.e., gated) by conformational reorientations at the surface interface. These results are later supported by structural studies using NMR and molecular modelling. New insights into conformational gating have also come from electrochemical experiments in which proteins are noncovalently adsorbed on the electrode surface. These systems have the advantage that it is relatively easy to vary systematically the driving force and electronic coupling. In this review we summarize the current knowledge obtained from these electrochemical experiments and compare it with some of the results obtained for protein-protein ET.     

Conformational reorganisation in interfacial protein electron transfer

Protein-protein electron transfer (ET) plays an essential role in all redox chains. Earlier studies which used cross-linking and increased solution viscosity indicated that the rate of many ET reactions is limited (i.e., gated) by conformational reorientations at the surface interface. These results are later supported by structural studies using NMR and molecular modelling. New insights into conformational gating have also come from electrochemical experiments in which proteins are noncovalently adsorbed on the electrode surface. These systems have the advantage that it is relatively easy to vary systematically the driving force and electronic coupling. In this review we summarize the current knowledge obtained from these electrochemical experiments and compare it with some of the results obtained for protein-protein ET.     

Effects of kinetic coupling on experimentally determined electron transfer parameters

Coupled electron transfer (ET) occurs when a relatively slow nonadiabatic ET reaction is preceded by a rapid but unfavorable adiabatic reaction that is required to activate the system for ET. As a consequence of this, the observed ET rate constant (k(ET)) is an apparent value equal to the product of the true k(ET) and the equilibrium constant for the preceding reaction step. Analysis of such reactions by ET theory may yield erroneous values for the reorganizational energy (lambda), electronic coupling (H(AB)), and ET distance that are associated with the true k(ET). If the DeltaG degrees dependence of the rate of a coupled ET reaction is analyzed, an accurate value of lambda will be obtained but the experimentally determined H(AB) will be less than the true H(AB) and the ET distance will be greater than the true distance. If the temperature dependence of the rate of a coupled ET reaction is analyzed, the experimentally determined value of lambda will be greater than the true lambda. The magnitude of this apparent lambda will depend on the magnitude of DeltaH degrees for the unfavorable reaction step that precedes ET. The experimentally determined values of H(AB) and distance will be accurate if DeltaS degrees for the preceding reaction is zero. If DeltaS degrees is positive, then H(AB) will be greater than the true value and the distance will be less than the true value. If DeltaS degrees is negative, then H(AB) will be less than the true value and the distance will be greater than the true value. Data sets for coupled ET reactions have been simulated and analyzed by ET theory to illustrate these points.     

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.     

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.     

Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.     

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers

From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of this process to < 50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. This change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.     

Rates and Equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse heme a --> CuA process were found to be 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.     

Effect of pressure on electron transfer reactions in inorganic and bioinorganic chemistry

Kinetic and thermodynamic studies involving the application of different high-pressure techniques, are very useful in gaining mechanistic information on the basis of volume changes that occur during inorganic and bioinorganic electron transfer reactions. The most fundamental type of electron transfer reaction concerns self-exchange reactions, for which the overall reaction volume is zero, and activation volumes can be measured and discussed. In the case of non-symmetrical electron transfer reactions, intra- and intermolecular processes can be studied and volume profiles can be constructed. Precursor complex formation can in some cases be recognized kinetically in such systems. Typical values of activation and reaction volumes are reviewed for various reversible and irreversible electron transfer reactions. Mechanistic conclusions reached on the basis of these parameters are presented. Volume profiles for electron transfer reactions enable a simplistic presentation of the reaction mechanism on the basis of intrinsic and solvational volume changes along the reaction coordinate.     

Correlation of electron transfer rate in photosynthetic reaction centers with intraprotein dielectric properties

A number of the electrogenic reactions in photosystem I, photosystem II, and bacterial reaction centers (RC) were comparatively analyzed, and the variation of the dielectric permittivity (epsilon) in the vicinity of electron carriers along the membrane normal was calculated. The value of epsilon was minimal at the core of the complexes and gradually increased towards the periphery. We found that the rate of electron transfer (ET) correlated with the value of the dielectric permittivity: the fastest primary ET reactions occur in the low-polarity core of the complexes within the picosecond time range, whereas slower secondary reactions take place at the high-polarity periphery of the complexes within micro- to millisecond time range. The observed correlation was quantitatively interpreted in the framework of the Marcus theory. We calculated the reorganization energy of ET carriers using their van der Waals volumes and experimentally determined epsilon values. The electronic coupling was calculated by the empirical Moser-Dutton rule for the distance-dependent electron tunneling rate in nonadiabatic ET reactions. We concluded that the local dielectric permittivity inferred from the electrometric measurements could be quantitatively used to estimate the rate constant of ET reactions in membrane proteins with resolved atomic structure with the accuracy of less than one order of magnitude.     

Correlation of electron transfer rate in photosynthetic reaction centers with intraprotein dielectric properties

A number of the electrogenic reactions in photosystem I, photosystem II, and bacterial reaction centers (RC) were comparatively analyzed, and the variation of the dielectric permittivity (ε) in the vicinity of electron carriers along the membrane normal was calculated. The value of ε was minimal at the core of the complexes and gradually increased towards the periphery. We found that the rate of electron transfer (ET) correlated with the value of the dielectric permittivity: the fastest primary ET reactions occur in the low-polarity core of the complexes within the picosecond time range, whereas slower secondary reactions take place at the high-polarity periphery of the complexes within micro- to millisecond time range. The observed correlation was quantitatively interpreted in the framework of the Marcus theory. We calculated the reorganization energy of ET carriers using their van der Waals volumes and experimentally determined ε values. The electronic coupling was calculated by the empirical Moser-Dutton rule for the distance-dependent electron tunneling rate in nonadiabatic ET reactions. We concluded that the local dielectric permittivity inferred from the electrometric measurements could be quantitatively used to estimate the rate constant of ET reactions in membrane proteins with resolved atomic structure with the accuracy of less than one order of magnitude.     

Electron transfer rates and equilibrium within cytochrome c oxidase.

Intramolecular electron transfer (ET) between the CuA center and heme a in bovine cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methyl nicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 830 nm. After the initial reduction phase, the 830 nm absorption was partially restored, corresponding to reoxidation of the CuA center. Concomitantly, the absorption at 445 nm and 605 nm increased, indicating reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration. This demonstrates that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse (heme a --> CuA) process were found to be 13 000 s-1 and 3700 s-1, respectively, at 25 degrees C and pH 7.4. This corresponds to an equilibrium constant of 3.4 under these conditions. Thermodynamic and activation parameters of the ET reactions were determined. The significance of these results, particularly the observed low activation barriers, are discussed within the framework of the known three-dimensional structure, ET pathways and reorganization energies.     

Electromagnetic acceleration of electron transfer reactions

The Moving Charge Interaction (MCI) model proposes that low frequency electromagnetic (EM) fields affect biochemical reactions through interaction with moving electrons. Thus, EM field activation of genes, and the synthesis of stress proteins, are initiated through EM field interaction with moving electrons in DNA. This idea is supported by studies showing that EM fields increase electron transfer rates in cytochrome oxidase. Also, in studies of the Na,K-ATPase reaction, estimates of the speed of the charges accelerated by EM fields suggest that they too are electrons. To demonstrate EM field effects on electron transfer in a simpler system, we have studied the classic oscillating Belousov--Zhabotinski (BZ) reaction. Under conditions where the BZ reaction oscillates at about 0.03 cycles/sec, a 60 Hz, 28 microT (280 mG) field accelerates the overall reaction. As observed in earlier studies, an increase in temperature accelerates the reaction and decreases the effect of EM fields on electron transfer. In all three reactions studied, EM fields accelerate electron transfer, and appear to compete with the intrinsic chemical forces driving the reactions. The MCI model provides a reasonable explanation of these observations.     

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.     

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein-DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein-DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor-operator DNA interaction, particularly O(R)1 and O(R)2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O(R)1 and O(R)2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O(R)1 DNA compared to O(R)2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299 nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein–DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein–DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor–operator DNA interaction, particularly O_R1 and O_R2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O_R1 and O_R2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O_R1 DNA compared to O_R2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299?nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

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.