alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.     

Measurement of the oxidation-reduction potentials for one-electron and two-electron reduction of electron-transfer flavoprotein from pig liver

Potentiometric titrations of pig liver electron-transfer flavoprotein (ETF) were performed at pH 7.5 and 4 degrees C, both in the reductive and oxidative directions. Reduction of ETF to the hydroquinone form required a total of two reducing equivalents/mol of ETF with the formation of sub-stoichiometric amounts of anionic semiquinone as an intermediate. The oxidation-reduction potentials for the two one-electron couples, oxidized ETF/ETF semiquinone and ETF semiquinone/fully reduced ETF, are +4 mV and -50 mV respectively. The overall midpoint potential for the two-electron couple (oxidized ETF/fully reduced ETF) is -23 mV.     

Extensive conformational sampling in a ternary electron transfer complex

Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.     

Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

Electron transfer flavoprotein from Methylophilus methylotrophus: properties, comparison with other electron transfer flavoproteins, and regulation of expression by carbon source.

When grown on methylated amines as a carbon source, Methylophilus methylotrophus synthesizes an electron transfer flavoprotein (ETF) which is the natural electron acceptor of trimethylamine dehydrogenase. It is composed of two dissimilar subunits of 38,000 and 42,000 daltons and 1 mol of flavin adenine dinucleotide. It was reduced by trimethylamine dehydrogenase to a stable anionic semiquinone form, which could not be converted, either enzymatically or chemically, to the fully reduced dihydroquinone. This ETF exhibited spectral properties which were nearly identical to ETFs from bacterium W3A1, Paracoccus denitrificans, and pig liver mitochondria. M. methylotrophus ETF cross-reacted immunologically and enzymatically with the ETF of bacterium W3A1 but not with the other two ETFs. In M. methylotrophus and bacterium W3A1, ETF and trimethylamine dehydrogenase were each expressed during growth on trimethylamine and were each absent during growth on methanol.     

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (相似文献   

X-ray scattering studies of Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein. Evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase

Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible "hinge" sequences that are unique to M. methylotrophus ETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an "induced fit" mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process.     

Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii catalyzes electron transfer from NADH or D-lactate dehydrogenase to butyryl-CoA dehydrogenase. As a basis for understanding the interactions of ETF with its substrates, we report here on the redox properties of ETF alone. ETF exhibited reversible, two-electron transfer during electrochemical reduction in the presence of mediator dyes. The midpoint redox potentials of the FAD cofactor were -0.185 V at pH 5.5, -0.259 V at pH 7.1 and -0.269 +/- 0.013 V at pH 8.4, all versus the standard hydrogen electrode In the presence of the indicator dye 1-deazariboflavin, the Nernst slopes were 0.029 V and 0.026 V at pH 5.5 and pH 7.1, respectively, compared with an expected value of 0.028 V at 10 degrees C. At pH 8.4, in the presence of 2-hydroxy-1,4-naphthoquinone or phenosafranine, the Nernst slope varied from 0.021 V to 0.041 V. In the experiments at pH 8.4, equilibration was very slow in the reductive direction and a difference of as much as 30 mV was observed between reductive and oxidative midpoints. ETF exhibited no thermodynamic stabilization of the radical form of the FAD cofactor during electrochemical reduction at pH 5.5, 7.1 or 8.4. However, up to 93% of kinetically stable, anionic radical was produced by dithionite titration at pH 8.5. Molar absorptivities of ETF radical were 17,000 M-1 X cm-1 at 365 nm and 5100 M-1 X cm-1 at 450 nm. The four ETF preparations used here contained less than 7% 6-OH-FAD. However, two of the preparations contained significant amounts (up to 30%) of flavin which stabilized radical and reduced at potentials 0.2 V more positive than those required for reduction of the major form of ETF. This is referred to as the B form of ETF. The proportion of ETF-FAD in the B form was increased by incubation with free FAD or by a cycle of reduction and reoxidation. These treatments caused marked changes in the absorption spectrum of oxidized ETF and decreases of 20-25% in ETF units/A450.     

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox properties of flavocytochrome c3 from Shewanella frigidimarina NCIMB400

The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.     

Formation of W(3)A(1) electron-transferring flavoprotein (ETF) hydroquinone in the trimethylamine dehydrogenase x ETF protein complex

The electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)) exhibits unusual oxidation-reduction properties and can only be reduced to the level of the semiquinone under most circumstances (including turnover with its physiological reductant, trimethylamine dehydrogenase (TMADH), or reaction with strong reducing reagents such as sodium dithionite). In the present study, we demonstrate that ETF can be reduced fully to its hydroquinone form both enzymatically and chemically when it is in complex with TMADH. Quantitative titration of the TMADH x ETF protein complex with sodium dithionite shows that a total of five electrons are taken up by the system, indicating that full reduction of ETF occurs within the complex. The results indicate that the oxidation-reduction properties of ETF are perturbed upon binding to TMADH, a conclusion further supported by the observation of a spectral change upon formation of the TMADH x ETF complex that is due to a change in the environment of the FAD of ETF. The results are discussed in the context of ETF undergoing a conformational change during formation of the TMADH x ETF electron transfer complex, which modulates the spectral and oxidation-reduction properties of ETF such that full reduction of the protein can take place.     

Electrochemical studies of arsenite oxidase: an unusual example of a highly cooperative two-electron molybdenum center

Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center. The reduction potential for the molybdenum center, 292 mV (vs SHE) at pH 5.9 and 0 degrees C, exhibits a linear pH dependence for pH 5-10, consistent with a two-electron reduction strongly coupled to the uptake of two protons without a pK in this range. This suggests that the oxidized enzyme is best characterized as having an L(2)MoO(2) rather than L(2)MoO(OH) center in the oxidized state and that arsenite oxidase uses a "spectator oxo" effect to facilitate the oxo transfer reaction. The onset of the catalytic wave observed in the presence of substrate correlates well with the Mo(VI/IV) potential, consistent with catalytic electron transport that is limited only by turnover at the active site. The one-electron peaks for the iron-sulfur centers are difficult to observe by protein film voltammetry, but spectrophotometric titrations have been carried out to measure their reduction potentials: at pH 6.0 and 20 degrees C, that of the [3Fe-4S] center is approximately 260 mV and that of the Rieske center is approximately 130 mV.     

Oxidation-reduction properties of glycolate oxidase

This is the first report of the redox potentials of glycolate oxidase. The pH dependence of the redox behavior as well as the effects of activators and inhibitors was studied. At pH 7.1 in 10 mM imidazole-chloride, Eo1' (EF1ox/EF1-.) is -0.033 +/- 0.010 V and Eo2' (EF1-./EF1redH-) is -0.017 +/- 0.017 V vs. the standard hydrogen electrode at 10 degrees C. A maximum of 29% flavin mononucleotide (FMN) anion radical is stabilized at half-reduction at pH 7.1 and 10 degrees C. Both redox couples of glycolate oxidase are pH-dependent from pH 7 to pH 9, and the FMN anion radical is stabilized in this range. The redox potentials of glycolate oxidase are shifted markedly positive of those of unbound FMN, consistent with the enzyme's function. The midpoint potential of glycolate oxidase is more positive than that of the glyoxalate/glycolate couple, and two-electron reduction of glycolate oxidase is thermodynamically favorable. The redox behavior of glycolate oxidase markedly contrasts that of other flavoprotein oxidases. For most flavoprotein oxidases, Eo1' is independent of pH from pH 7 to pH 9 and is much more positive than Eo2', which is pH-dependent. We present a mechanism that suggests a structural basis for the positive shifts and pH dependence of both Eo1' and Eo2' of glycolate oxidase.     

Reactions of electron-transfer flavoprotein and electron-transfer flavoprotein: ubiquinone oxidoreductase.

Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-Q oxidoreductase) catalyses the re-oxidation of reduced electron-transfer flavoprotein (ETF) with ubiquinone-1 (Q-1) as the electron acceptor. A kinetic assay for the enzyme was devised in which glutaryl-CoA in the presence of glutaryl-CoA dehydrogenase was used to reduce ETFox. and the reduction of Q-1 was monitored at 275 nm. The partial reactions involved in the overall assay system were examined. Glutaryl-CoA dehydrogenase catalyses the rapid reduction of ETFox. to the anionic semiquinone (ETF.-), but reduces ETF.- to the fully reduced form (ETFhq) at a rate that is about 6-fold lower. ETF.-, but not ETFhq, is directly re-oxidized by Q-1 at a rate that, depending on the steady-state concentration of ETF.-, may contribute significantly to the overall reaction. ETF-Q oxidoreductase catalyses rapid disproportionation of ETF.- with an equilibrium constant of about 1.0 at pH 7.8. In the presence of Q-1 it also catalyses the re-oxidation of ETFhq at a rate that is faster than that of the overall reaction. Rapid-scan experiments indicated the formation of ETF.-, but its fractional concentration in the early stages of the re-oxidation of ETFhq is low. The data indicate that the re-oxidation of ETFhq proceeds at a rate that is adequate to account for the overall rate of electron transfer from glutaryl-CoA to Q-1. An unusual property of ETF-Q oxidoreductase seems to be that it not only catalyses the re-oxidation of the reduced forms of ETF but also facilitates the complete reduction of ETFox. to ETFhq by disproportionation of the radical.     

The interaction of trimethylamine dehydrogenase and electron-transferring flavoprotein

The interaction between the physiological electron transfer partners trimethylamine dehydrogenase (TMADH) and electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus has been examined with particular regard to the proposal that the former protein "imprints" a conformational change on the latter. The results indicate that the absorbance change previously attributed to changes in the environment of the FAD of ETF upon binding to TMADH is instead caused by electron transfer from partially reduced, as-isolated TMADH to ETF. Prior treatment of the as-isolated enzyme with the oxidant ferricenium essentially abolishes the observed spectral change. Further, when the semiquinone form of ETF is used instead of the oxidized form, the mirror image of the spectral change seen with as-isolated TMADH and oxidized ETF is observed. This is attributable to a small amount of electron transfer in the reverse of the physiological direction. Kinetic determination of the dissociation constant and limiting rate constant for electron transfer within the complex of (reduced) TMADH with (oxidized) ETF is reconfirmed and discussed in the context of a recently proposed model for the interaction between the two proteins that involves "structural imprinting" of ETF.     

Electron-transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) was purified to homogeneity from pig liver submitochondrial particles. It is comparable in molecular weight and general properties to ETF-QO from beef heart [Ruzicka, F. J., & Beinert, H. (1977) J. Biol. Chem. 252, 8440-8445], and the electron spin resonance signals of the reduced iron-sulfur cluster are essentially identical. ETF-QO catalyzes the transfer of electrons from electron-transfer flavoprotein (ETF) to nitro blue tetrazolium, with a sluggish reaction turnover number of about 10-30 min-1. In contrast, the enzyme rapidly disproportionates ETF semiquinone, with a turnover number of 200 s-1. The reverse reaction, comproportionation of oxidized and hydroquinone ETF, provides an enzymatic assay for ETF-QO with picomolar sensitivity. Equilibrium spectrophotometric titrations show that ETF-QO accepts a maximum of two electrons from ETF and accepts three electron equivalents from dithionite or by photochemical reduction. All electrons from the enzymatically or chemically reduced protein can be transferred to 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (PB), and this reaction is readily reversible. Reduction of ETF-QO by 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzohydroquinone is pH dependent and indicates the enzyme to have a redox potential that decreases by 47 mV per pH unit. Therefore, ETF-QO binds one to two protons upon reduction. The EO' at pH 7.3 is 38 mV. The ability of ETF-QO to catalyze the equilibration of ETF redox states has been used to evaluate the equilibrium 2ETFsq + nH+ in equilibrium ETFox + ETFhq.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of oxidation-reduction potentials of anthranilate hydroxylase from Trichosporon cutaneum by substrate and effector binding

The pH dependence of the redox behavior of anthranilate hydroxylase from Trichosporon cutaneum in its uncomplexed and anthranilate-complexed forms, as well as the effects on the reduction potential, at pH 7.4, of enzyme in complex with 3-methylanthranilate, salicylate, 3-acetylpyridine adenine dinucleotide phosphates, and azide plus anthranilate, is described. At pH 7.4 the midpoint potential of uncomplexed enzyme (EFlox/EFlredH-) is -0.229 V vs SHE, close to that of free flavin. The aromatic substrates and effector all shift the midpoint potential value in a positive direction by 0.068-0.100 V. This shift results in thermodynamically more favorable reduction of the substrate/effector-complexed enzyme by NADPH. Consistent with thermodynamic considerations, the aromatic substrates (or effector) are bound to the reduced enzyme 2-4 orders of magnitude more tightly than to the oxidized enzyme. The tighter binding of the substrate to the two-electron-reduced enzyme may be related to the double hydroxylation reaction performed by this enzyme, which is a more complex reaction than is carried out by typical flavoprotein hydroxylases. The acetylpyridine nucleotides appear to have no significant regulatory role.     

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.     

The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.     

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.