Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

Engineering the respiratory complex I to energy-converting NADPH:ubiquinone oxidoreductase

The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.     

Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase

The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.     

Differences between the reactivities of two pyridine nucleotides in the rapid reduction process and the reoxidation process of adrenodoxin reductase.

The reaction process of adrenodoxin reductase with NADPH and NADH were investigated. The appearance of new intermediate with a broad absorption band at around 520 nm has been detected by rapid-scan stopped-flow spectrophotometry. Although the formation of this intermediate is more rapid with NADPH than with NADH, the rates of the subsequent decay to the fully reduced state are almost identical (Kobs values were 20.5 and 16.0s-1). These results indicate that the new intermediate is the complex formed between the oxidized enzyme and reduced pyridine nucleotide (enzyme-substrate complex), and that subsequent decay of the intermidiate is caused by a two-electron transfer process from the reduced pyridine nucleotide to the enzyme flavin. On the other hand, spectral and kinetic properties in the steady state of the reoxidation reaction of the enzyme reduced with NADPH and NADH were somewhat different. The rate of reoxidation of the enzyme under aerobic conditions from the reduced state to the oxidized state was 6.5 times faster when a 10-fold molar excess of NADH was used than when NADPH of the same concentration was used. This result is consistent with the fact that the NADH-dependent oxidase activity was 6.4 times greater than that dependent on NADPH. During reoxidation of the reduced enzyme under aerobic conditions in the presence of an excess of NADPH or NADH, the EPR spectra indicated the formation of the flavin semiquinone radical species. Similarly, the formation of semiquinone was observed in the absorption spectrum with either NADPH or NADH under the same conditions as in the EPR measurement. The intensity of the semiquinone signal on EPR was considerably smaller with NADH than with NADPH. These results suggest that NADP+ complex with the enzyme semiquinone protects the radical from oxidation by oxygen to a greater extent than NAD+, and consequently the semiquinone is easier to detect with NADPH than with NADH.     

The reduction of putidaredoxin reductase by reduced pyridine nucleotides

Putidaredoxin reductase (PdR), an FAD-containing protein, mediates the transfer of electrons from NADH to putidaredoxin in the cytochrome P-450cam-dependent oxidation of camphor. Using stopped-flow spectrophotometry, reduction of putidaredoxin reductase by NADH (70 microM) at 4 degrees C appeared to be a pseudo-first-order process with a rate constant in excess of 600 s-1. The reduction of putidaredoxin reductase by NADPH was much slower with a second-order rate constant of 530 s-1 M-1 at 4 degrees C. The reduction of the enzyme was monitored at several wavelengths: 455 nm to follow flavin reduction; 700 nm to follow the appearance of the long-wavelength charge-transfer complex; and 513 nm to detect the presence of a semiquinone form of the flavoprotein. There was no apparent semiquinone formation observed during reduction. The charge-transfer complex can be formed in the presence of NAD+, whereas, no charge-transfer band could be detected when PdR was reduced with NADPH. The titration of chemically or NADPH-reduced putidaredoxin reductase with either a stoichiometric or an excess amount of NAD+ resulted in the formation of a charge-transfer complex, indicating that the reduced form of PdR has a high affinity for NAD+ regardless of the method of reduction. The data presented indicate that putidaredoxin reductase is reduced without the formation of semiquinone intermediate and, upon reduction, forms a tight complex with NAD+. The Keq for the reduction of PdR by NADPH is 1.1 and the midpoint potential for this reaction is -317 +/- 5 mV.     

Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a contributor to cellular oxidative stress. In isolated complex I the reduced flavin is known to react with molecular oxygen to form predominantly superoxide, but studies using intact mitochondria contend that superoxide may result from a semiquinone species that responds to the proton-motive force (Δp) also. Here, we use bovine heart submitochondrial particles to show that a single mechanism describes superoxide production by complex I under all conditions (during both NADH oxidation and reverse electron transfer). NADH-induced superoxide production is inhibited by complex I flavin-site inhibitors but not by inhibitors of ubiquinone reduction, and it is independent of Δp. Reverse electron transfer (RET) through complex I in submitochondrial particles, driven by succinate oxidation and the Δp created by ATP hydrolysis, reduces the flavin, leading to NAD(+) and O(2) reduction. RET-induced superoxide production is inhibited by both flavin-site and ubiquinone-reduction inhibitors. The potential dependence of NADH-induced superoxide production (set by the NAD(+) potential) matches that of RET-induced superoxide production (set by the succinate potential and Δp), and they both match the potential dependence of the flavin. Therefore, both NADH- and RET-induced superoxide are produced by the flavin, according to the same molecular mechanism. The unified mechanism describes how reactive oxygen species production by complex I responds to changes in cellular conditions. It establishes a route to understanding causative connections between the enzyme and its pathological effects and to developing rational strategies for addressing them.     

The NAD(P)H:flavin oxidoreductase from Escherichia coli. Evidence for a new mode of binding for reduced pyridine nucleotides.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2'-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.     

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

Geobacter sulfurreducens strain PCA oxidized acetate to CO2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases NADP+ and NAD+, respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and strictly CoA-dependent; a low activity was also observed with NADP+. Succinate dehydrogenase and fumarate ductase both were membrane-bound. Succinate oxidation was coupled to NADP+ reduction whereas fumarate reduction was coupled to NADPH and NADH Coupling of succinate oxidation to NADP+ or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.     

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.     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

The rotenone-insensitive reduction of quinones and nitrocompounds by mitochondrial NADH:ubiquinone reductase

The rotenone-insensitive reduction of quinones and aromatic nitrocompounds by mitochondrial NADH: ubiquinone reductase (complex I, EC 1.6.99.3) has been studied. It was found that these reactions proceed via a mixed one- and two-electron transfer. The logarithms of the bimolecular rate constants of oxidation (TN/Km) are proportional to the one-electron-reduction potentials of oxidizers. The reactivities of nitrocompounds are close to those of quinones. Unlike the reduction of ferricyanide, these reactions are not inhibited by NADH. However, they are inhibited by NAD+ and ADP-ribose, which also act as the mixed-type inhibitors for ferricyanide. TN/Km of quinones and nitrocompounds depend on the NAD+/NADH ratio, but not on NAD+ concentration. They are diminished by the limiting factors of 2.5-3.5 at NAD+/NADH greater than 200. It seems that rotenone-insensitive reduction of quinones and nitrocompounds takes place near the NAD+/NADH and ferricyanide binding site, and the inhibition is caused by induced conformational changes after the binding of NAD+ or ADP-ribose.     

The flavoprotein subcomplex of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria: insights into the mechanisms of NADH oxidation and NAD+ reduction from protein film voltammetry

Complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria contains 45 different subunits and nine redox cofactors. NADH is oxidized by a noncovalently bound flavin mononucleotide (FMN), then seven iron-sulfur clusters transfer the two electrons to quinone, and four protons are pumped across the inner mitochondrial membrane. Here, we use protein film voltammetry to investigate the mechanisms of NADH oxidation and NAD+ reduction in the simplest catalytically active subcomplex of complex I, the flavoprotein (Fp) subcomplex. The Fp subcomplex was prepared using chromatography and contained the 51 and 24 kDa subunits, the FMN, one [4Fe-4S] cluster, and one [2Fe-2S] cluster. The reduction potential of the FMN in the enzyme's active site is lower than that of free FMN (thus, the oxidized state of the FMN is most strongly bound) and close to the reduction potential of NAD+. Consequently, the catalytic transformation is reversible. Electrocatalytic NADH oxidation by subcomplex Fp can be explained by a model comprising substrate mass transport, the Michaelis-Menten equation, and interfacial electron transfer kinetics. The difference between the "catalytic" potential and the FMN potential suggests that the flavin is reoxidized before NAD+ is released or that intramolecular electron transfer from the flavin to the [4Fe-4S] cluster influences the catalytic rate. NAD+ reduction displays a marked activity maximum, below which the catalytic rate decreases sharply as the driving force increases. Two possible models reproduce the observed catalytic waveshapes: one describing an effect from reducing the proximal [2Fe-2S] cluster and the other the enhanced catalytic ability of the semiflavin state.     

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.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.     

The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart.

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.     

A ternary mechanism for NADH oxidation by positively charged electron acceptors, catalyzed at the flavin site in respiratory complex I

The flavin mononucleotide in complex I (NADH:ubiquinone oxidoreductase) catalyzes NADH oxidation, O(2) reduction to superoxide, and the reduction of several 'artificial' electron acceptors. Here, we show that the positively-charged electron acceptors paraquat and hexaammineruthenium(III) react with the nucleotide-bound reduced flavin in complex I, by an unusual ternary mechanism. NADH, ATP, ADP and ADP-ribose stimulate the reactions, indicating that the positively-charged acceptors interact with their negatively-charged phosphates. Our mechanism for paraquat reduction defines a new mechanism for superoxide production by complex I (by redox cycling); in contrast to direct O(2) reduction the rate is stimulated, not inhibited, by high NADH concentrations.     

Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.     

A catalytically active complex formed from the recombinant dI protein of Rhodospirillum rubrum transhydrogenase, and the recombinant dIII protein of the human enzyme

Transhydrogenase is a proton pump. It has three components: dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively, and dII spans the membrane. We have expressed dIII from Homo sapiens transhydrogenase (hsdIII) in Escherichia coli. The purified protein was associated with stoichiometric amounts of NADP(H) bound to the catalytic site. The NADP+ and NADPH were released only slowly from the protein, supporting the suggestion that nucleotide-binding by dIII is regulated by the membrane-spanning dII. HsdIII formed a catalytically active complex with recombinant dI from Rhodospirillum rubrum (rrdI), even in the absence of dII. The rates of forward and reverse transhydrogenation catalysed by this complex are probably limited by slow release from dIII of NADPH and NADP+, respectively. The hybrid complex also catalysed high rates of 'cyclic' transhydrogenation, indicating that hydride transfer, and exchange of nucleotides with dI, are rapid. Stopped-flow experiments revealed a rapid, monoexponential, single-turnover burst of reverse transhydrogenation in pre-steady-state. The apparent first-order rate constant of the burst increased with the concentration of rrdI. A deuterium isotope effect (kH/kD approximately 2 at 27 degrees C) was observed when [4B-1H]NADPH was replaced with [4B-2H]NADPH. The characteristics of the burst of transhydrogenation with rrdI:hsdIII differed from those previously reported for rrdI:rrdIII (J.D. Venning et al., Eur. J. Biochem. 257 (1998) 202-209), but the differences are readily explained by a greater dissociation constant of the hybrid complex. The steady-state rate of reverse transhydrogenation by the rrdI:hsdIII complex was almost independent of pH, but there was a single apparent pKa ( approximately 9.1) associated with the cyclic reaction. The reactions of the dI:dIII complex probably proceed independently of those protonation/deprotonation reactions which, in the complete enzyme, are associated with H+ translocation.