Regulation of carcinogen metabolism in the rat ovary by the estrous cycle and gonadotropin.

The activity of 7,12-dimethylbenz[a]anthracene hydroxylase in the rat ovary is several times higher in the proestrous phase of the estrous cycle than in the estrous and metestrous plus diestrous phases. Administration of gonadotropin leads to a similar increase in the capacity of the ovary to metabolize xenobiotics. This variation in the activity of 7,12-dimethylbenz[a]anthracene hydroxylase during the estrous cycle may be related to the marked changes in the incidence of ovarian cancer during menopause and in women taking contraceptive pills.

Metabolism of polycyclic aromatic hydrocarbons and covalent binding of metabolites to protein in rat adrenal gland.

The metabolism of the carcinogenic and adrenocorticolytic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene in rat adrenals was investigated. Both 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene, which also is a well-known carcinogen but has no short-term effects on rat adrenals, appear to be metabolized by one common type of cytochrome P-450-dependent monooxygenase localized in the endoplasmic reticulum. Studies of the kinetic properties of this cytochrome P-450 reveal that the Km values for 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene are lower than 3 microM. Identification of metabolites indicates that, with both 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene, phenols and diols were formed the relative rates of formation of which were markedly influenced by the expoxide hydrase inhibitor cyclohexane oxide, suggesting that epoxides are intermediate metabolites. Added or endogenous microsomal glutathione S-transferase B had little or no effect on the distribution of metabolites. A rather selective binding of metabolites of 7,12-dimethylbenz(a)anthracene to soluble and microsomal proteins was demonstrated. The adrenal cytochrome P-450 involved in the conversion of these polycyclic aromatic hydrocarbons appears to be unrelated to those responsible for the synthesis of mineralocorticoids and glucocorticoids from cholesterol. Among androgens and estrogens, estradiol proved to be the most inhibitory steroid, suggesting a role of the hydrocarbon-metabolizing cytochrome P-450 in estrogen biosynthesis. However, no such function could be demonstrated conclusively. The metabolite patterns and the effects of nonsteroid inhibitors of liver monooxygenases, e.g., alpha-naphthoflavone, SU 9055, and ellipticine, suggest that the properties of this cytochrome P-450 resemble those of the 3-methyl-cholanthrene-inducible cytochrome P-488 from rat liver.

Selective solubilization of the components of the mitochondrial inner membrane by lysolecithin.

1. Of various phospholipids tested, lysolecithin was the most efficient in the solubilization of the components of beef heart submitochondrial particles. Lysolecithin solubilized selectively nicotinamide nucleotide transhydrogenase, succinate dehydrogenase, NADH dehydrogenase and oligomycin-sensitive ATPase. Various cytochromes other than cytochrome c were only slightly solubilized. 2. The effect of various parameters, e.g. ionic strength, pH, time of centrifugation, and concentrations of lysolecithin and protein was investigated. Increasing times of centrifugation led to a partial sedimentation of NADH dehydrogenase, and a complete sedimentation of oligomycin-sensitive ATPase and cytochrome oxidase. 3. Further fractionation of the lysolecithin extract by centrifugation in the presence of low concentrations of cholate gave a complete separation of NADH dehydrogenase and transhydrogenase, indicating that these enzymes are not related functionally. 4. With the lysolecithin fractionation procedure a more than 10-fold purification of transhydrogenase was achieved. Polyacrylamide gel electrophoresis of the partially purified transhydrogenase in the presence of sodium dodecyl sulphate showed major increases in protein-stained bands corresponding to between 70 000 and 54 000 daltons. 5. A possible mechanism for the detergent action of lysolecithin involving a specific exchange of bound phospholipids for lysolecithin is discussed.

Interactions of reduced and oxidized nicotinamide mononucleotide with wild-type and alphaD195E mutant proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli.

The interaction of reduced nicotinamide mononucleotide (NMNH), constituting one half of NADH, with the wild-type and alphaD195E proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated. Reduction of thio-NADP+ by NMNH was catalysed at approximately 30% of the rate with NADH. Other activities including proton pumping and the cyclic reduction of 3'-acetyl-pyridine-NAD+ by NMNH in the presence of NADP+ were more strongly inhibited. The alphaD195 residue is assumed to interact with the 2'-OH moiety of the adenosine-5'-phosphate, i.e., the second nucleotide of NADH. Mutation of this residue to alphaD195E resulted in a 90% decrease in activity with NMNH as well as NADH as substrate, suggesting that it produced global structural changes of the NAD(H) binding site. The results suggest that the NMN moiety of NADH is a substrate of transhydrogenase, and that the adenine nucleotide is not required for catalysis or proton pumping.

The transmembrane domain and the proton channel in proton-pumping transhydrogenases.

Proton-pumping nicotinamide nucleotide transhydrogenases are composed of three main domains, the NAD(H)-binding and NADP(H)-binding hydrophilic domains I (dI) and III (dIII), respectively, and the hydrophobic domain II (dII) containing the assumed proton channel. dII in the Escherichia coli enzyme has recently been characterised with regard to topology and a packing model of the helix bundle in dII is proposed. Extensive mutagenesis of conserved charged residues of this domain showed that important residues are betaHis91 and betaAsn222. The pH dependence of betaH91D, as well as betaH91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD(+) by NADPH, i.e., the reverse reaction, is optimal at a pH essentially coinciding with the pK(a) of the residue in the beta91 position. It is therefore concluded that the wild-type transhydrogenase is regulated by the degree of protonation of betaHis91. The mechanisms of the interactions between dI+dIII and dII are suggested to involve pronounced conformational changes in a 'hinge' region around betaR265.

The cDNA sequence of proton-pumping nicotinamide nucleotide transhydrogenase from man and mouse.

cDNA clones for the human and mouse nicotinamide nucleotide transhydrogenases have been isolated and their sequences have been determined. Multiple alignments show that the functional proteins are encoded by single mRNAs. The deduced amino acid sequences are approximately 95% identical for the previously known bovine, and the human and mouse proteins. The major variable region is located in the presequence. It is proposed that all mammalian transhydrogenases have a similar structure.

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.

Comparative studies on nicotinamide nucleotide transhydrogenase from different sources.

(1) Nicotinamide nucleotide transhydrogenases in submitochondrial particles from beef heart mitochondria, chromatophores from Rhodospirillum rubrum and membrane preparations from Escherichia coli and Pseudomonas aeruginosa have been compared with respect to the following properties: stereospecificity for the 4-hydrogen of NADH, reactivity with 3'-NADP, inhibition by palmityl-CoA, sensitivity tot rypsin, and effects of Ca2+ and 2'-AMP on the reaction rates. (2) Transhydrogenases from submitochondrial particles, R. rubrum chromatophores and E. coli membrane preparations have A-side stereospecificity for NADH, do not react with 3'-NADP, are inhibited by palmityl-CoA and are extremely sensitive to trypsin treatment. No effects of Ca2+ or 2'-AMP on the reaction rates were observed. In R. rubrum chromatophores trypsin-sensitive sites are present both in the soluble transhydrogenase factor and in the membrane preparation devoid of transhydrogenase factor. (3) In contrast, P. aeruginosa transhydrogenase is allosterically regulated by Ca2+ and 2'-AMP, is reactive with 3'-NADP, has B-side stereospecificity for NADH and is insensitive to palmityl-CoA or trypsin treatment. (4) It is concluded that the properties characterizing the transhydrogenase in E. coli, R. rubrum chromatophores and submitochondrial particles are closely connected with the interaction of the enzyme with an energy-conserving membrane system.

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase.

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids. 2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition. 3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols. 4. It is concluded that mitochondrial transhydrogenase, catalyzing the nonenergy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.

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.

Effects of metal ions on the substrate-specificity and activity of proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli.

Nicotinamide nucleotide transhydrogenase catalyzes the reversible reduction of NADP+ by NADH and a concomitant proton translocation. It was demonstrated (Glavas, N.A. and Bragg, P.D. (1995) Biochim. Biophys. Acta 1231, 297-303) that the Escherichia coli transhydrogenase also catalyzed a reduction of the NAD-analogue 3-acetylpyridine-NAD+ (AcPyAD+) by NADH at low pH and in the absence of (added) NADP(H) and high salt concentrations The mechanism of this reaction has as yet not been explained. In the present study, the E. coli transhydrogenase was purified by affinity chromatography through the NADP(H)-site, rendering the pure enzyme free of NADP(H). Using this preparation it was confirmed that the enzyme readily catalyzes the above reaction. Inhibitors specific for the NADP(H)-site, e.g., palmitoyl-Coenzyme A and adenosine-2'-monophosphate-5'-diphosphoribose, strongly inhibited the reduction of AcPyAD+ by NADH, whereas an inhibitor of the NAD(H)-site, adenosine 5'-diphosphoribose, was less inhibitory. This suggests that a lack of metal ions or other ions at low pH induces an unspecific interaction of the NADP(H)-site with AcPyAD+ or NADH, presumably NADH, producing a cyclic reduction of AcPyAD+ by NADH via NAD(H) bound in the NADP(H) site. A stimulation of reduction of AcPyAD+ by NADPH by Mg2+ present during reconstitution of transhydrogenase in phospholipid vesicles was observed, but it is presently unclear whether this effect is related to that seen with the detergent-dispersed enzyme.

Purification and initial characterization of microsomal epoxide hydrolase from the human adrenal gland.

Microsomal epoxide hydrolase from the human adrenal gland was purified to a high degree of homogeneity in 10% overall yield using sequential chromatography on DE-52, FPLC Mono Q and FPLC Superose columns. The fact that the overall purification was only 7.3-fold indicates that approx. 14% of the total microsomal protein consisted of this enzyme, a uniquely high value. The human adrenal enzyme was found to resemble rat liver microsomal epoxide hydrolase closely in a number of respects, including molecular weight, N-terminal amino-acid sequence and response to low-molecular weight ligands. However, rabbit antibodies directed against human adrenal microsomal epoxide hydrolase crossreacted only weakly with the corresponding rat liver protein. The relatively high levels of microsomal epoxide hydrolase in the human adrenal gland suggest that this enzyme may be of particular importance in this tissue. However, very little cytochrome P-450-catalyzed metabolism of xenobiotics has been demonstrated in the human adrenal and our present results speak against the involvement of microsomal epoxide hydrolase in the steroid metabolism of this gland. Thus, the function of this enzyme in the human adrenal is enigmatic.

NBD-Cl modification of essential residues in mitochondrial nicotinamide nucleotide transhydrogenase from bovine heart.

Modification of mitochondrial nicotinamide nucleotide transhydrogenase (NADPH: NAD+ oxidoreductase, EC 1.6.1.1) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), followed by measurement of the absorption or fluorescence of the transhydrogenase-NBD adducts, resulted in a biphasic labelling of approx. 4-6 sulfhydryls, presumably cysteine residues. Of these 1-2 (27%) were fast-reacting and 3-4 (73%) slow-reacting sulfhydryls. In the presence of substrates, e.g., NADPH, the labelling was monophasic and all sulfhydryls were fast-reacting, suggesting that the modified sulfhydryls are predominantly localized peripheral to the NAD(P)(H)-binding sites. The rates of modification allowed the calculation of the rate constants for each phase of the labelling. Both in the absence and in the presence of a substrate, e.g., NADPH, the extent of labelling essentially parallelled the inhibition of transhydrogenase activity. Attempts to reactivate transhydrogenase by reduction of labelled sulfhydryls were not successful. Photo-induced transfer of the NBD adduct in partially inhibited transhydrogenase, from the sulfhydryls to reactive NH2 groups of amino-acid residue(s), identified as lysine residue(s), was parallelled by an inhibition of the residual transhydrogenase activity. It is suggested that a lysine localized close to the fast-reacting NBD-Cl-reactive sulfhydryl groups is essential for activity.

Characterization of the interaction of NADH with proton pumping E. coli transhydrogenase reconstituted in the absence and in the presence of bacteriorhodopsin.

(1) Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was purified in a reconstitutively active form employing affinity chromatography on immobilized palmitoyl-Coenzyme A. Reconstituted transhydrogenase showed an active proton pumping and a stimulation of the rate of reduction of 3-acetylpyridine-NAD+ by NADPH by uncouplers. Reconstitution in the absence of a thiol-reducing agent, e.g. dithiothreitol, abolished proton pumping without affecting catalytic activity, giving a decoupled transhydrogenase. (2) Co-reconstitution of transhydrogenase with bacteriorhodopsin gave vesicles which catalyzed a 5-10-fold increased rate of reduction of thio-NADP+ by NADH in the light. The Km for NADH, but not that for thio-NADP+, decreased markedly in the light, indicating an effect of the electrochemical proton potential on the affinity of the enzyme for NADH. Inhibition by substrate derivatives in the absence or presence of light supported this conclusion. Replacement of NADH with 2'-deoxy-NADH gave a strongly sigmoidal concentration dependence, indicating an allosteric change induced by binding to the NAD(H)-site. (3) Reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, previously demonstrated to be catalyzed by both reconstituted bovine transhydrogenase and detergent-dispersed E. coli transhydrogenase, occurred at a pH below 6.5. This reaction did not pump protons. Proton pumping by 3-acetylpyridine-NAD+ plus NADPH occurred at a pH above 5.5. The two reactions were thus close to mutually exclusive, with a cross point at pH 5.8. Assuming a helix bundle structure of the membrane domain of transhydrogenase, a model is proposed involving histidine 91 of the beta subunit which previously was shown to be essential by site-directed mutagenesis. According to the model the extent of protonation of this histidine determines whether proton pumping or the NADH-3-acetylpyridine-NAD+ reaction takes place.

Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli.

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

Characterization of a nicotinamide nucleotide transhydrogenase gene from the green alga Acetabularia acetabulum and comparison of its structure with those of the corresponding genes in mouse and Caenorhabditis elegans.

Proton-pumping nicotinamide nucleotide transhydrogenase (Nnt) is a membrane-bound enzyme that catalyzes the reversible reduction of NADP(+) by NADH. This reaction is linked to proton translocation across the membrane. Depending on metabolic conditions, the enzyme may be involved in NADPH generation, e.g., for detoxification of peroxides and/or free radicals and protection from ischemic damage. Nnt exists in most prokaryotes and in animal mitochondria. It is composed of 2-3 subunits in bacteria and of a single polypeptide in mitochondria. An open question is whether Nnt exists in any photosynthetic eukaryotes and if so, to which class it belongs. In the present study it is demonstrated that, by cloning and sequencing cDNA and genomic copies of its NNT gene, an ancient alga, Acetabularia acetabulum (Chlorophyta, Dasycladales), contains a nuclear-encoded Nnt. In contrast to photosynthetic bacteria, this algal Nnt is composed of a single polypeptide of the class found in animal mitochondria. Excluding a poly(A) tail, NNT cDNA from A. acetabulum is 3688 bp long, consists of eight exons and spans 17 kb. The NNT gene from mouse was also characterized. Subsequently, the gene organization of the A. acetabulum NNT was compared to those of the homologous mouse (100 kb and 21 exons) and Caenorhabditis elegans (5.1 kb and 18 exons) genes.

ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles.

The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD+ were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)--an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276-287)--when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD+ required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the delta mu H+. In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al.

Properties of a proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli with alpha and beta subunits linked through fused transmembrane helices.

Proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of an alpha and a beta subunit, whereas the homologues mitochondrial enzyme contains a single polypeptide. As compared to the latter transhydrogenase, using a 14-helix model for its membrane topology, the point of fusion is between the transmembrane helices 4 and 6 where the fusion linker provides the extra transmembrane helix 5. In order to clarify the potential role of this extra helix/linker, the alpha and the beta subunits were fused using three connecting peptides of different lengths, one (pAX9) involving essentially a direct coupling, a second (pKM) with a linking peptide of 18 residues, and a third (pKMII) with a linking peptide of 32 residues, as compared to the mitochondrial extra peptide of 27 residues. The results demonstrate that the plasma membrane-bound and purified pAX9 enzyme with the short linker was partly misfolded and strongly inhibited with regard to both catalytic activities and proton translocation, whereas the properties of pKM and pKMII with longer linkers were similar to those of wild-type E. coli transhydrogenase but partly different from those of the mitochondrial enzyme although pKMII generally gave higher activities. It is concluded that a mitochondrial-like linking peptide is required for proper folding and activity of the E. coli fused transhydrogenase, and that differences between the catalytic properties of the E. coli and the mitochondrial enzymes are unrelated to the linking peptide. This is the first time that larger subunits of a membrane protein with multiple transmembrane helices have been fused with retained activity.

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties.

Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.

Domains, specific residues and conformational states involved in hydride ion transfer and proton pumping by nicotinamide nucleotide transhydrogenase from Escherichia coli.

Nicotinamide nucleotide transhydrogenase constitutes a proton pump which links the NAD(H) and NADP(H) pools in the cell by catalyzing a reversible reduction of NADP+ by NADH. The recent cloning and characterization of several proton-pumping transhydrogenases show that they share a number of features. They are composed of three domains, i.e., the hydrophilic domains I and III containing the NAD(H)- and NADP(H)-binding sites, respectively, and domain II containing the transmembrane and proton-conducting region. When expressed separately, the two hydrophilic domains interact directly and catalyze hydride transfer reactions similar to those catalyzed by the wild-type enzyme. An extensive mutagenesis program has established several amino acid residues as important for both catalysis and proton pumping. Conformational changes mediating the redox-driven proton pumping by the enzyme are being characterized. With the cloned, well-characterized and easily accessible transhydrogenases from E. coli and Rhodospirillum rubrum at hand, the overall aim of the transhydrogenase research, the understanding of the conformationally driven proton pumping mechanism, is within reach.