An analysis of some thermodynamic properties of iron-sulphur centres in site I of mitochondria.

1. The midpoint potentials of the various iron-sulphur centres in Site I were determined at different pH values by the technique of redox potentiometry. An interesting feature is the pH-dependence of Centre N-2, the highest potential component of the NADH dehydrogenase segment of the respiratory chain. 2. The apparent midpoint potentials of Centre N-2 (NADH dehydrogenase) and S-1 (succinate dehydrogenase) and their pH-dependence was also determined by using the succinate/fumarate couple. Again Centre N-2 is pH-dependent in midpoint potential, and Centre S-1 is not. The results obtained by titrating with the succinate/fumarate couple are in quantitative agreement with those obtained for these centres by redox potentiometry. 3. Oxidation-reduction titrations of iron-sulphur centres with the couple NADH/NAD+ and an analogue APADH/APAD+ in the presence of rotenone gave results substantially different from those obtained by redox potentiometry; these differences may be due to the mechanism of action of NADH dehydrogenase and its specific interaction with NADH. 5. The addition of ATP to an NAD+/NADH-poised system induces an uncoupler-sensitive oxidation of Centre N-4.     

Flow-force relationships during energy transfer between mitochondrial proton pumps.

The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, delta mu H+, and output flows of the ATPase, redox and transhydrogenase H(+)-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, delta mu H+, in an extended range of delta mu H+, independently of whether the H(+)-generating pump is the ATPase or a redox pump, or whether delta mu H+ is depressed by inhibitors of the H(+)-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H(+)-pumps exhibit a steep dependence on delta mu H+. The flow-force relationships differ depending on whether the depression of delta mu H+ is induced by inhibitors of the H(+)-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H(+)-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4-. With redox site I as proton-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4-. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H(+)-pumps.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Kinetics of NADH oxidation of NAD+ reduction by mitochondrial complex I]

The kinetics of the NAD: artificial acceptor-oxidoreductase and delta mu H(+)-dependent succinate: NAD(+)-oxidoreductase reactions (reverse electron transfer) reactions catalyzed by the membrane-bound complex I was studied. The values of apparent rate constants of dissociation of complexes of the oxidized and reduced enzyme with NAD+ and NADH were determined. It was shown that the apparent affinity of NADH for the oxidized complex I is by nearly three orders of magnitude as high as that of the reduced one; a reverse correlation is found for NAD+. A kinetic scheme of complex I functioning in the forward and reverse reactions, according to which the free reduced enzyme is not an intermediate of the forward (NADH-oxidase) reaction and the free oxidized enzyme is not an intermediate of the reverse (NAD(+)-reductase) reaction, is proposed.     

Coupling site I and the rotenone-sensitive ubisemiquinone in tightly coupled submitochondrial particles

The rotenone-sensitive g = 2.00 low temperature EPR signal attributed to ubisemiquinone is observed in submitochondrial particles during coupled electron transfer from NADH to oxygen and from succinate to NAD+. The signal is seen only in the presence of oligomycin added to induce the respiratory control (7-9 with NADH and 3-4 with succinate) and it disappears in the presence of uncouplers (CCCP or gramicidin D). No reduction of the iron-sulfur center N-2 in the presence of 20 mM succinate and cyanide is observed, thus suggesting that N-2 is not in equilibrium with the ubiquinone pool. A hypothesis is proposed on delta mu H+ generation coupled with electron transfer between iron-sulfur center N-2 and the ubiquinone pool.     

Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent

The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.     

Control of cellular redox potential as measured in a steady-state, cell-free system

A cell-free system consisting of rat liver mitochondria, liver cytosol, lactate, and the substrates intrinsic to the malate-aspartate shuttle was reconstituted for studies of steady-state substrate fluxes and, more specifically, to evaluate further the mechanism of control of the intra- and extramitochondrial steady states of the free NAD+/NADH ratios. Soluble (F1) ATPase or 2,4-dinitrophenol (DNP) were added in varying amounts to alter substrate fluxes and the constant energy state of this 'open' metabolizing system. The steady-state redox segregation (1.36 log NAD+/NADH ratio out vs NAD+/NADH in the mitochondrial matrix) was maximally about 3 kcal, and declined together with the membrane potential (delta psi) and log ATP/ADP, which obtain on imposing an increasing energy load on the system. It is concluded that transmembrane movement of reducing equivalents is coupled to electron transfer through delta psi, mediated by the electrogenic exchange of glutamate and aspartate. When delta psi was high (near State 4), delta G redox was approximately the same as that generated without flux of reducing equivalents [E. J. Davis, J. Bremer, and K. E. Akerman (1980) J. Biol. Chem. 255, 2277-2283], suggesting that delta Gredox is in near thermodynamic equilibrium with delta psi. If the steady-state ATP/ADP ratio was altered with an energy load (F1-ATPase), delta Gredox decreased more steeply than delta psi (tetraphenyl phosphonium-sensitive electrode used to measure delta psi). At comparable ranges of ATP/ADP, both delta Gredox and delta psi decreased more steeply with uncoupler than with an external ADP-regenerating system.     

Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 +/- 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- --> PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 +/- 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.     

Energetics of ATP-driven reverse electron transfer from cytochrome c to fumarate and from succinate to NAD in submitochondrial particles

The maximum Gibbs free energies of reverse electron transfer from succinate to NAD+ and from cytochrome c to fumarate driven by ATP hydrolysis in submitochondrial particles from beef heart were measured as a function of the Gibbs free energy of ATP hydrolysis. The ratio of the energies delta G'redox/delta G'ATP was 1.40 from succinate to NAD+ and 0.89 from cytochrome c to succinate. The ratio, equivalent to a thermodynamic P/2e-ratio, was dependent on whether the electrochemical proton gradient was primarily a membrane potential or a pH gradient for the cytochrome c to fumarate reaction. The results are consistent with H+/ATP = 3 for F1 ATPase, H+/2e- = 4 for NADH-CoQ reductase, and H+(matrix)/2e- = 2 for succinate-cytochrome c reductase.     

The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force

Evidence is presented for a proportional relationship between the extramitochondrial phosphate potential (delta Gexp) and the proton-motive force (delta mu H+) across the mitochondrial membrane in rat-liver mitochondria oxidising succinate in State 4, when delta mu H+ is varied by addition of uncouplers or malonate. This relationship was found when precautions were taken to minimise interference with the determination of delta Gexp and delta mu H+ by intramitochondrial nucleotides, adenylate kinase activity, the quenching method, and delta mu H+-dependent changes in matrix volume. A non-proportional delta Gexp/delta mu H+ relationship was obtained when these precautions were omitted. Our results do not support mosaic protonic coupling, but are not necessarily in conflict with other localised coupling schemes.     

Interaction of ATPase from submitochondrial fragments and a natural inhibitor protein during delta-mu-H+ generation on a membrane

An addition of the inhibitor protein (IF1) to submitochondrial particles (SMP) essentially free of endogenous IF1 (AS-SMP) results in a synchroneous inhibition of ATP hydrolysis and ATP-dependent reduction of NAD+ by succinate without any effect on the oxidative phosphorylation rate. The binding of IF1 to the membrane-bound ATPase leads to the loss of the inhibitor protein sensitivity to trypsin despite the delta mu H+ generation. The data obtained are consistent with a model according to which there exist the hydrolase and synthetase forms of F1 and contradict the generally accepted concepts on the delta mu H+-dependent dissociation of the F1-IF1 complex.     

Studies on the mechanism of oxidative phosphorylation. Flow-force relationships in mitochondrial energy-linked reactions

The relationship between the steady-state level of membrane potential (delta psi) and the rates of energy production and consumption has been studied in mitochondria and submitochondrial particles. The energy-linked reactions investigated were oxidative phosphorylation (with NADH, succinate, and beta-hydroxybutyrate as respiratory substrates) and nucleoside triphosphate-driven transhydrogenation from NADH to NADP and uphill electron transfer from succinate to NAD. Results have shown the following. 1) Attenuation of the rates of the energy-producing reactions results in a parallel change in the rates of the energy-consuming reactions with little or no change in the magnitude of steady-state delta psi. 2) At low rates of energy production and consumption, steady-state delta psi decreases. However, this is due largely to the energy leak of the system which lowers static-head delta psi when the rate of energy production is slow. 3) When the rate of energy production and static-head delta psi are held constant, and the rate of energy consumption is diminished by partial inhibition or the use of suboptimal conditions (e.g. subsaturating substrate concentrations), then even a small decrease in the rate of energy consumption results in an upward adjustment of the level of steady-state delta psi. The lower the rate of energy input, the greater the upward adjustment of steady-state delta psi upon suppression of the rate of energy consumption. 4) The above results have been discussed with regard to the role of bulk-phase delta mu H+ or delta psi in the mitochondrial energy transfer reactions.     

Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

A hypothesis for the role of dithiol-disulfide interchange in solute transport and energy-transducing processes

We have recently shown that the physical mechanism for delta approximately mu H+-driven changes in the Km for three different transport systems is an oxidation-reduction reaction involving a dithiol-disulfide interconversion [Robillard, G.T. and Konings, W.N. (1981) Biochemistry, 20, 5025-5032; Konings, W.N. and Robillard, G.T. (1982) Proc. Natl Acad. Sci. USA, in the press]. Based on the similarities between the data from these three systems and published data from other systems, we now propose that dithiol-disulfide interchange may play a general role in membrane-related processes such as transport, energy transduction and hormone-receptor interactions. We propose that the affinities of the substrate-binding sites are regulated by a dithiol and a disulfide situated at different depths in the membrane. In addition we propose that the oxidation states of these two redox centers are coupled by dithiol-disulfide interchange such that, when one is oxidized, the other is reduced. Since a transmembrane electrical potential, delta psi, or a pH gradient, delta pH, can alter the redox state, it can change the affinity of the substrate-binding sites. The delta approximately mu H+-induced changes in affinity are sufficient to drive active transport (symport or antiport) and energy-transducing processes. A similar mechanism can be applied to transport systems driven by phosphorylated enzyme intermediates instead of delta approximately mu H+. Changes of the redox potential in a given compartment during metabolism could also control the affinity of ligand binding even in the absence of a delta approximately mu H+. The ligand-binding affinities of facilitated diffusion transport systems and receptor proteins may be regulated in this manner.     

Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.     

DCCD sensitivity of electron and proton transfer by NADH: ubiquinone oxidoreductase in bovine heart submitochondrial particles--a thermodynamic approach

The sensitivity of the H+/2e- ratio of the redox-driven proton pumping by the NADH: ubiquinone reductase (complex I) of the submitochondrial particles to dicyclohexylcarbodiimide (DCCD) was studied by a thermodynamic approach, measuring the membrane potential and delta pH across the membrane and the redox potential difference across the complex I span of the respiratory chain. The delta Gr/delta muH+ ratio did not decrease upon additions of 50 or 100 nmol of DCCD per mg protein in the presence of oligomycin although the H+/2e- ratio has been demonstrated to decrease upon DCCD addition in kinetic experiments with mitochondria. Complex I then becomes reminiscent of the cytochrome bc1 complex, which shows DCCD sensitivity of the kinetically but not thermodynamically determined H+/2e- ratio.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles

The in vitro translocation of OmpF-Lpp, a model secretory protein, into inverted membrane vesicles of Escherichia coli obligatorily requires the proton motive force (delta mu H+) in the conventional assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The translocation, however, took place efficiently, even in the absence of delta mu H+, when the system was supplemented with additional SecA. With the stripped membrane vesicles, which are permeable to protons, or in the absence of NADH, the supplementation of SecA remarkably stimulated the translocation activity. The further addition of NADH did not significantly enhance the translocation activity under the SecA-enriched conditions. OmpF-Lpp thus translocated could be recovered from the vesicular lumen by sonication, indicating that complete translocation occurred in the absence of delta mu H+. It is suggested that delta mu H+ is required for high affinity interaction of SecA with the presumed secretory machinery in the cytoplasmic membrane and that a high concentration of SecA modulates the delta mu H+ requirement.