Reaction of electron-transfer flavoprotein with electron-transfer flavoprotein-ubiquinone oxidoreductase

The oxidative half-reaction of electron-transfer flavoprotein (ETF), electron transfer from ETF to electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), is dependent on complementary surface charges on the two proteins. ETF is the positively charged member of the redox pair. The evidence is based on the pH and ionic strength dependencies of the comproportionation of oxidized ETF and ETF hydroquinone catalyzed by ETF-QO and on the effects of chemical modification of ETF on the comproportionation reaction. Acetylation of one and five epsilon-amino groups of lysyl residues results in 3- and 13-fold increases, respectively, in the Km of ETF-QO for ETF but no change in Vmax. Amidination, which maintains positive charge at modified loci, has no effect on steady-state kinetic constants. These chemical modifications have no effect on the equilibrium constant for equilibration of ETF redox states. The Km of ETF-QO for ETF is pH dependent above pH 8.5, suggesting titration of lysyl residues as previously observed in studies of the reductive half-reaction of ETF [Beckmann, J. D., & Frerman, F. E. (1983) J. Biol. Chem. 258, 7563-7569]. The ionic strength dependence of TN/KmETF for the reaction follows the limiting Br?nsted equation ln (TN/Km) = ln k0 + 2 alpha Z1Z2I1/2, and Z1Z2, the product of charges on the reacting proteins, is similar to the value of Z1Z2 for the reductive half-reaction of ETF by the general acyl-CoA dehydrogenase. The ETF-QO-catalyzed comproportionation reaction exhibits a primary deuterium isotope effect in D2O, perhaps indicating the participation of solvent water in the electron-transfer reaction.     

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.     

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO)

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.     

Reaction of electron-transfer flavoprotein ubiquinone oxidoreductase with the mitochondrial respiratory chain

Submitochondrial particles catalyze the reduction of electron-transfer flavoprotein (ETF) by NADH and succinate under anaerobic conditions in reactions that are totally inhibited by rotenone and thenoyl trifluoroacetone, respectively. The particles also catalyze the ATP-dependent reduction of NAD+ by enzymatically reduced ETF. The latter reaction is inhibited by rotenone and carbonyl cyanide chlorophenylhydrazone and all three reactions are inhibited by antibody to electrontransfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). These observations indicated that ETF-QO reacts with the pool of ubiquinone that is reduced by NADH and succinic dehydrogenases. Consistent with this hypothesis, NADH- and succinic-ETF reductase activities are inhibited 99% in ubiquinone-depleted particles, and reincorporation of exogenous ubiquinone restores at least 90% of these activities. Reduction of the bc1 complex by ETF and acyl CoA oxidase activity are also inhibited by antibody to ETF-QO. Myxothiazole and antimycin which inhibit the quinonol oxidation and quinone reduction sites, respectively, in the bc1 complex also inhibit electron transport from ETF-QO through the complex according to current models of the Q-cycle (Rich, P.R. (1986) J. Bioenerg. Biomembranes 18, 145-156). The results show that ETF-QO is an obligatory component of the electron transport pathway between ETF and the ubiquinone pool and suggest a mechanism for the steady-state turnover of ETF-QO.     

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   

Inhibition of naphthohydroquinone autoxidation by DT-diaphorase (NAD(P)H:[quinone acceptor] oxidoreductase)

Summary

It has been reported that little redox cycling occurs during the reduction of 2-methyl-1,4-naphthoquinone by DT-diaphorase, suggesting that the reduction product, 2-methyl-1,4-naphthohydroquinone, does not readily undergo autoxidation. In the present study, however, it has been shown that DT-diaphorase, by virtue of its ability to re-reduce the naphthoquinone formed in the oxidation reaction, decreases the rate of autoxidation of 2-methyl-1,4- naphthohydroquinone. Therefore, the low rate of redox cycling observed does not reflect an intrinsic stability of the hydroquinone but inhibition of its autoxidation by the enzyme. Redox cycling of 2,3-dimethyl-, 2,3-dimethoxy- and 2-methoxy-1,4-naphthoquinone, and the autoxidation of their respective hydroquinones, were similarly inhibited by diaphorase. The concentration of the enzyme required for inhibition varied widely among the different compounds, and this was related to the autoxidation rate of the hydroquinone and the rate at which the corresponding quinone was reduced by diaphorase. The behaviour of 2-hydroxy-1,4-naphthoquinone was exceptional in that the rate of redox cycling increased with increasing levels of diaphorase and no inhibition of the autoxidation of the hydroquinone derived from this substance could be demonstrated, even at very high enzyme concentrations. The results of the present experiments indicate that the relative stability of naphthohydroquinones cannot be judged on the basis of studies involving reduction of the quinone by DT-diaphorase and suggest that current concepts on the role of this enzyme in the detoxification of quinones may need revision.     

The electron transfer flavoprotein: ubiquinone oxidoreductases

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).     

Concerted action of DT-diaphorase and superoxide dismutase in preventing redox cycling of naphthoquinones: An evaluation

It has been suggested that the enzymes DT-diaphorase and superoxide dismutase act in concert to prevent redox cycling of naphthoquinones and thus protect against the toxic effects of such substances. Little is known, however, about the scope of this process or the conditions necessary for its operation. In the presence of low levels of DT-diaphorase, 2-methyl-1,4-naphthoquinone was found to undergo redox cycling. This was very effectively inhibited by SOD, and in the presence of both enzymes the hydroquinone was maintained in the reduced form. The inhibitory effect of the enzyme combination was overcome, however, at high concentrations of the quinone, or by small increases in pH. Furthermore, redox cycling was re-established by addition of haemoproteins such as cytochrome c and methaemoglobin. DT-diaphorase and SOD strongly inhibited redox cycling of 2,3-dimethyl- and 2,3-dimethoxy-1,4-naphthoquinone, but not that of 2-hydroxy-, 5-hydroxy- or 2-amino-1,4-naphthoquinone. Inhibition of redox cycling by a combination of DT-diaphorase and SOD is therefore not applicable to all naphthoquinone derivatives, and when it does occur, it may be overwhelmed at high quinone concentrations, and it may not operate under slightly alkaline conditions or in the presence of tissue components capable of initiating hydroquinone autoxidation.     

Quinone-enhanced ascorbate reduction of nitric oxide: role of quinone redox potential

The quinones 1,4-naphthoquinone (NQ), methyl-1,4-naphthoquinone (MNQ), trimethyl-1,4-benzoquinone (TMQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ-0) enhance the rate of nitric oxide (NO) reduction by ascorbate in nitrogen-saturated phosphate buffer (pH 7.4). The observed rate constants for this reaction were determined to be 16±2,215±6,290±14 and 462±18 M^-1 s^-1, for MNQ, TMQ, NQ and UQ-0, respectively. These rate constants increase with an increase in quinone one-electron redox potential at neutral pH, E₇¹. Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.     

2,3-Dimethoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4-naphthoquinones: glycation inhibitors with lipid peroxidation activity

Anti-glycation activity of our anti-oxidant quinone library was measured and several 2,3-dimethoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4-naphthoquinones were identified as novel inhibitors of glycation, of which 2,3-dimethoxy-5-methyl-1,4-benzoquinones 13b is the most potent glycation inhibitor with around 50 microM of the IC(50) value.     

Quinone-enhanced Ascorbate Reduction of Nitric Oxide: Role of Quinone Redox Potential

The quinones 1,4-naphthoquinone (NQ), methyl-1,4-naphthoquinone (MNQ), trimethyl-1,4-benzoquinone (TMQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ-0) enhance the rate of nitric oxide (NO) reduction by ascorbate in nitrogen-saturated phosphate buffer (pH 7.4). The observed rate constants for this reaction were determined to be 16±2,215±6,290±14 and 462±18?M^-1?s^-1, for MNQ, TMQ, NQ and UQ-0, respectively. These rate constants increase with an increase in quinone one-electron redox potential at neutral pH, E₇₁. Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.     

Interaction of electron acceptors with thylakoids from halophytic and non-halophytic species

Thylakoid membranes isolated from halophytic species showed differences in their interactions with ionic and lipophilic electron acceptors when compared to thylakoids from non-halophytes. FeCN was considerably less efficient as electron acceptor with halophyte thylakoids, supporting much lower rates of O₂ evolution and having a lower affinity. FeCN accepted electrons at a different, DMMIB insensitive, site with these thylakoids. 1,4-Benzo-quinones with less positive midpoint potentials were less effective in accepting electrons from halophyte thylakoids compared to nonhalophyte thylakoids, also reflected in lower rates of O₂ evolution and lower affinity. Considering the lipolphilic nature and the fact that there was no apparent change in the site donating electrons to the quinones, an alteration in the midpoint potential of this site by about +100mV is postulated for the halophyte thylakoids.Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - Cyt b₆/f cytochrome b₆/f complex - DBMIB 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone - DCBQ 2,6-dichloro-1,4-benzoquinone - DCIP 2,6-dichlorophenol-indolphenol - DMBQ 2,5-dimethyl-1,4-benzoquinone - E_m7 midpoint redox potential at pH 7.0, FeCN-K₃Fe(CN)₆ - HNQ 5-hydroxy-1,4-naphthoquinone - MV methylviologen - NQ 1,4-naphthoquinone - PBQ phenyl-1,4-benzoquinone - PC plastocyanin - PQ plastoquinone     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H(2)O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately -240 mV) and low-spin (approximately -110, -190 and -265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (E(m) = -80 mV) in the presence of NADH (E(m) = -320 mV) and an NADH-menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.     

Synthesis and biological activity of derivatives of ubiquinone: photoaffinity analogues containing the 4-azido-2-nitroanilino group

The photoaffinity analogues of ubiquinone 2,3-dimethoxy-5-methyl-6-[2-[1-oxo-3-(4-azido-2-nitroanilino) propoxy]-3-methylbutyl]-1,4-benzoquinone (2'-ANAP-Q-1) and 2,3-dimethoxy-5-methyl-6-[3-[1-oxo-3-(4-azido-2-nitroanilino) propoxy]-3-methylbutyl]-1,4-benzoquinone (3'-ANAP-Q-1) have been synthesized. The required intermediate alcohols 2,3-dimethoxy-5-methyl-6-(2-hydroxy-3-methylbutyl)-1,4-benzoquinone and 2,3-dimethoxy-5-methyl-6-(3-hydroxy-3-methylbutyl)-1,4-benzoquinone were prepared in good yield from ubiquinone 1 by hydration of the side-chain double bond via hydroboration or acid catalysis, respectively. These alcohols were then coupled with 3-(4-azido-2-nitroanilino)propanoic acid, with p-toluenesulfonyl chloride in dry pyridine, to give 2'- and 3'-ANAP-Q-1. The synthetic methods presented should be of general utility in the preparation of derivatives of ubiquinone in which a reactive or reporter group is relatively close to the ubiquinone ring. By use of membrane vesicles prepared from a ubi-men-strain of Escherichia coli described previously [Wallace, B., & Young, I. G. (1977) Biochim. Biophys. Acta 461, 84-100], it has been shown that 2'- and 3'-ANAP-Q-1 substitute for ubiquinone 8 in the NADH, succinate, and D-lactate oxidase systems. Thus, these compounds may be of value in labeling respiratory chain proteins that interact with ubiquinone.     

Redox potentials and kinetic properties of fumarate reductase complex from Vibrio succinogenes

The isolated menaquinol: fumarate oxidoreductase (fumarate reductase complex) from Vibrio succinogenes was investigated with respect to the redox potentials and the kinetic response of the prosthetic groups. The following results were obtained. (1) The redox state of the components was measured as a function of the redox potential established by the fumarate/succinate couple, after freezing of the samples (173 K). From these measurements, the midpoint potential of the [2Fe-2S] cluster (−59 mV), the [4Fe-4S] cluster (−24 mV) and the flavin/flavosemiquinone couple (about −20 mV) was obtained. (2) Potentiometric titration of the enzyme in the presence of electron-mediating chemicals gave, after freezing, apparent midpoint potentials that were 30–100 mV more negative than those found with the fumarate/succinate couple. (3) The rate constants of reduction of the components on the addition of succinate or 2,3-dimethyl-1,4-naphthoquinol were as great as or greater than the corresponding turnover numbers of the enzyme in quinone reduction by succinate or fumarate reduction by the quinol. In the oxidation of the reduced enzyme by fumarate, cytochrome b oxidation was about as fast as the corresponding turnover number of quinol oxidation by fumarate, while the [2Fe-2S] and half of the [4Fe-4S] cluster responded more than 2-times slower. The rate constant of the other half of the 4-Fe cluster was one order of magnitude smaller than the turnover number.     

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

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.