trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components.

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E. coli transhydrogenase.

The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266. A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values. It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.

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.

Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli. Effects on catalytic and H+-pumping activities.

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase. Despite harsh treatments with, e.g. detergents and denaturing agents, the alpha and beta subunits remained tightly associated. A monoclonal antibody directed towards the alpha subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II. Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.

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.

Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants.

Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated. Out of 16 charged residues studied, 6 have not been previously investigated. The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH. This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH. These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91. The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site. The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction. However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding. beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site. The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered.

Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. A study of structure and function.

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H). A model defining betaA, alphaB, betaB, betaD, and betaE of this domain is presented. To test this model, four single cysteine mutants (cfbetaA348C, cfbetaA390C, cfbetaK424C, and cfbetaR425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined. The results are consistent with the view that, in agreement with the model, beta-Ala348, beta-Arg350, beta-Ala390, beta-Asp392, and beta-Lys424 are located in or close to the NADP(H) site. More specifically, beta-Ala348 succeeds betaB. The remarkable reactivity of betaR350C toward NNADP suggests that this residue is close to the nicotinamide moiety of NADP(H). beta-Ala390 and beta-Asp392 terminate or succeed betaD, and are thus, together with the region following betaA, creating the switch point crevice where NADP(H) binds. beta-Asp392 is particularly important for the substrate affinity, but it could also have a more complex role in the coupling mechanism for transhydrogenase.

Catalytic properties of hybrid complexes of the NAD(H)-binding and NADP(H)-binding domains of the proton-translocating transhydrogenases from Escherichia coli and Rhodospirillum rubrum.

Transhydrogenase couples reversible hydride transfer from NADH to NADP+ to proton translocation across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. The enzyme is composed of three parts. Domain I (dI) and domain III (dIII) are water soluble and contain the binding sites for NAD(H) and NADP(H), respectively; domain II (dII) spans the membrane. In the present investigation, dI from Rhodospirillum rubrum (rrI) and Escherichia coli (ecI), and dIII from R. rubrum (rrIII) and E. coli (ecIII) were overexpressed in E. coli and subsequently purified. Also, a preparation of a partially degraded E. coli transhydrogenase (ecbeta) was examined. Catalytic activities were analyzed in various dI+dIII and dI+ecbeta combinations. The abilities of the different dI+dIII combinations to catalyze cyclic transhydrogenation, i.e., the reduction of AcPyAD+ by NADH mediated via tightly bound NADP(H) in dIII, varied in the order: rrI+ecIII approximately rrI+rrIII > rrI+ecbeta >> ecI+ecIII; no measurable activities for ecI+rrIII and ecI+ecbeta were detected. Thus, rrI has a much greater apparent affinity than ecI for ecIII or rrIII or ecbeta. The pH dependences of the cyclic reaction seem to be determined by scalar protonation events on dI, both in rrI+rrIII and ecI+ecIII mixtures as well as in the wild-type R. rubrum and possibly in the E. coli enzyme. Higher reverse activities for rrI+ecbeta than for rrI+ecIII confirmed the regulatory role of dII for the association and dissociation rates of NADP(H).

Stopped-flow kinetics of hydride transfer between nucleotides by recombinant domains of proton-translocating transhydrogenase.

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP+ (k approximately 0.03 s(-1)). Phase A of the burst (k approximately 600 s(-1)) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k approximately 10-50 s(-1)), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B-2H]NADPH, and by [4B-1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27535-27538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I:III complex.

Mutation of amino acid residues in the mobile loop region of the NAD(H)-binding domain of proton-translocating transhydrogenase.

The effects of single amino acid substitutions in the mobile loop region of the recombinant NAD(H)-binding domain (dI) of transhydrogenase have been examined. The mutations lead to clear assignments of well-defined resonances in one-dimensional 1H-NMR spectra. As with the wild-type protein, addition of NADH, or higher concentrations of NAD+, led to broadening and some shifting of the well-defined resonances. With many of the mutant dI proteins more nucleotide was required for these effects than with wild-type protein. Binding constants of the mutant proteins for NADH were determined by equilibrium dialysis and, where possible, by NMR. Generally, amino acid changes in the mobile loop region gave rise to a 2-4-fold increase in the dI-nucleotide dissociation constants, but substitution of Ala236 for Gly had a 10-fold effect. The mutant dI proteins were reconstituted with dI-depleted bacterial membranes with apparent docking affinities that were indistinguishable from that of wild-type protein. In the reconstituted system, most of the mutants were more inhibited in their capacity to perform cyclic transhydrogenation (reduction of acetyl pyridine adenine dinucleotide, AcPdAD+, by NADH in the presence of NADP+) than in either the simple reduction of AcPdAD+ by NADPH, or the light-driven reduction of thio-NADP+ by NADH, which suggests that they are impaired at the hydride transfer step. A cross-peak in the 1H-1H nuclear Overhauser enhancement spectrum of a mixture of wild-type dI and NADH was assigned to an interaction between the A8 proton of the nucleotide and the betaCH3 protons of Ala236. It is proposed that, following nucleotide binding, the mobile loop folds down on to the surface of the dI protein, and that contacts, especially from Tyr235 in a Gly-Tyr-Ala motif with the adenosine moiety of the nucleotide, set the position of the nicotinamide ring of NADH close to that of NADP+ in dIII to effect direct hydride transfer.

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.

Interdomain hydride transfer in proton-translocating transhydrogenase.

We describe the use of the recombinant, nucleotide-binding domains (domains I and III) of transhydrogenase to study structural, functional and dynamic features of the protein that are important in hydride transfer and proton translocation. Experiments on the transient state kinetics of the reaction show that hydride transfer takes place extremely rapidly in the recombinant domain I:III complex, even in the absence of the membrane-spanning domain II. We develop the view that proton translocation through domain II is coupled to changes in the binding characteristics of NADP+ and NADPH in domain III. A mobile loop region which emanates from the surface of domain I, and which interacts with NAD+ and NADH during nucleotide binding has been studied by NMR spectroscopy and site-directed mutagenesis. An important role for the loop region in the process of hydride transfer is revealed.

The pH dependences of reactions catalyzed by the complete proton-translocating transhydrogenase from Rhodospirillum rubrum, and by the complex formed from its recombinant nucleotide-binding domains.

Transhydrogenase couples the translocation of protons across a membrane to the transfer of reducing equivalents between NAD(H) and NADP(H). Using transhydrogenase from Rhodospirillum rubrum we have examined the pH dependences of the 'forward' and 'reverse' reactions, and of the 'cyclic' reaction (NADP(H)-dependent reduction of the analogue, acetyl pyridine adenine dinucleotide, by NADH). In the case of the membrane-bound protein in chromatophores, the imposition of a protonmotive force through the action of the light-driven electron-transport system, stimulated forward transhydrogenation, inhibited reverse transhydrogenation, but had no effect on the cyclic reaction. The differential response at a range of pH values provides evidence that hydride transfer per se is not coupled to proton translocation and supports the view that energy transduction occurs at the level of NADP(H) binding. Chromatophore transhydrogenase and the detergent-dispersed enzyme both have bell-shaped pH dependences for forward and reverse transhydrogenation. The cyclic reaction, however, is rapid at low and neutral pH, and is attenuated only at high pH. A mixture of recombinant purified NAD(H)-binding domain I, and NADP(H)-binding domain III, of R. rubrum transhydrogenase carry out the cyclic reaction with a similar pH profile to that of the complete enzyme, but the forward and reverse reactions were much less pH dependent. The rates of release of NADP+ and of NADPH from isolated domain III were pH independent. The results are consistent with a model for transhydrogenation, in which proton binding from one side of the membrane is consequent upon the binding of NADP+ to the enzyme, and then proton release on the other side of the membrane precedes NADPH release.

Role of methionine-239, an amino acid residue in the mobile-loop region of the NADH-binding domain (domain I) of proton-translocating transhydrogenase.

Transhydrogenase couples the transfer of hydride equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The one-dimensional proton NMR spectrum of the recombinant NAD(H)-binding domain (domain I) of transhydrogenase from Rhodospirillum rubrum reveals well-defined resonances, several of which arise from a mobile loop at the protein surface. Four have been assigned to Met residues (MetA-MetD). Substitution of Met239 with either Ile (dI.M239I) or Phe (dI.M239F) resulted in loss of MetA from the NMR spectrum. Broadening and shifting of the mobile loop resonances consequent on NAD(H) binding indicate that the loop closes down on the protein surface. More NAD(H) had to be added to mutant domain I than to wild type to give comparable resonance broadening. The Kd of domain I for NADH, measured by equilibrium dialysis, was increased about three-fold by the Met239 mutations. Mutant and wild-type domain I were reconstituted with domain I-depleted membranes from R. rubrum, and with recombinant domain III of transhydrogenase. With membranes, the Km for acetylpyridine adenine dinucleotide during reverse transhydrogenation was 5x and > 6x greater in dI.M239I and dI.M239F, respectively, than in wild-type. Cyclic transhydrogenation (in membranes and the recombinant system) was substantially more inhibited (70% in dI.M239I, and 84% in dI.M239F) than either forward or reverse transhydrogenation. The docking affinities of dI.M239I and dI.M239F to the depleted membranes were similar to those of wild-type. It is concluded that Met239 is MetA in the mobile loop of domain I, and that in proteins with amino acid substitutions at this position, the binding affinity of NAD(H) is decreased, and the hydride transfer step is inhibited.

Evidence that the transfer of hydride ion equivalents between nucleotides by proton-translocating transhydrogenase is direct.

The molecular masses of the purified, recombinant nucleotide-binding domains (domains I and III) of transhydrogenase from Rhodospirillum rubrum were determined by electrospray mass spectrometry. The values obtained, 40,273 and 21,469 Da, for domains I and III, respectively, are similar to those estimated from the amino acid sequences of the proteins. Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleotides bound to these proteins. The transient-state kinetics of hydride transfer catalyzed by mixtures of recombinant domains I and III were studied by stopped-flow spectrophotometry. The data indicate that oxidation of NADPH, bound to domain III, and reduction of acetylpyridine adenine dinucleotide (an NAD+ analogue), bound to domain I, are simultaneous and very fast. The transient-state reaction proceeds as a biphasic burst of hydride transfer before establishment of a steady state, which is limited by slow release of NADP+. Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation.

Mutations at tyrosine-235 in the mobile loop region of domain I protein of transhydrogenase from Rhodospirillum rubrum strongly inhibit hydride transfer.

Transhydrogenase from mitochondrial and bacterial membranes couples proton translocation to hydride transfer between NAD(H) and NADP(H). The enzyme has three domains, of which domains I and III protrude from the membrane. These possess the NAD(H)- and NADP(H)-binding sites, respectively, whereas domain II spans the membrane. In domain I there is a mobile loop which emanates from the surface of the protein, but which closes down upon NAD(H) binding. In this report we show that the NADP(H)-dependent reduction of acetylpyridine adenine dinucleotide by NADH catalysed by Rhodospirillum rubrum transhydrogenase has 'ping-pong' kinetics, confirming that the reaction is cyclic. We then describe the kinetic and thermodynamic properties of mutants of recombinant domain I protein from the R. rubrum enzyme, in which Tyr-235 in the mobile loop has been substituted with Phe or Asn residues (dI.Y235F and dI.Y235N, respectively). (1) Equilibrium dialysis measurements show that dI.Y235F and dI.Y235N bind NADH more weakly than wild-type domain I protein (the Kd increases twofold and fourfold, respectively). (2) Reverse transhydrogenation rates (in steady state) of domain I-depleted membrane vesicles reconstituted with either dI.Y235F or dI.Y235N are inhibited by about 50% and 78%, respectively, relative to those obtained in reconstitutions with wild-type domain I protein. (3) Reverse transhydrogenation rates (in steady state) of mixtures of recombinant domain III protein and either dI.Y235F or dI.Y235N are inhibited only by about 10% and 20%, respectively, relative to those obtained in mixtures with wild-type protein. (4) Forward transhydrogenation rates (in both the complete enzyme and in domain I:III complexes) are inhibited even less by the mutations than the reverse reactions. (5) In contrast with (1), (2) and (3), cyclic transhydrogenation was strongly inhibited in both the reconstituted membrane system and in the recombinant domain I:III complexes (only 7-8% activity remains with dI.Y235F, and only 2-3% with dI.Y235N). It was recently established that, in contrast to forward and reverse transhydrogenation, the cyclic reaction is substantially limited by the rate of hydride transfer. It is therefore concluded that mutations at Tyr-235 in the mobile loop severely disrupt the hydride transfer step in the catalytic reaction of transhydrogenase.

The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact?

Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.

Properties of the purified, recombinant, NADP(H)-binding domain III of the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum.

Transhydrogenase comprises three domains. Domains I and III are peripheral to the membrane and possess the NAD(H)- and NADP(H)-binding sites, respectively, and domain II spans the membrane. Domain III of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified. The purified protein was associated with substoichiometric quantities of tightly bound NADP+ and NADPH. Fluorescence spectra of the domain III protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close. The rate constants for NADP+ release and NADPH release from domain III were 0.03 s-1 and 5.6 x 10(4) s-1, respectively. In the absence of domain II a mixture of the recombinant domain III protein, plus the previously described recombinant domain I protein, catalysed reduction of acetylpyridine-adenine dinucleotide (AcPdAD+) by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP+ from domain III. Similarly, the mixture catalysed reduction of thio-NADP+ by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain III. The mixture also catalysed very rapid reduction of AcPdAD+ by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain III and vice versa indicated (a) that during reduction of AcPdAD+ by NADPH, a single domain I protein can visit and transfer H equivalents to about 60 domain III proteins during the time taken for a single domain III to release its NADP+, whereas (b) the cyclic reaction is rapid on the timescale of formation and break-down of the domain I. III complex. The rate of the hydride transfer reaction was similar in the domain I.III complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I.III complex due to the greatly decreased rates of release of NADP+ and NADPH. It is concluded that, in the complete enzyme, conformational changes in the membrane-spanning domain II, which result from proton translocation, lead to changes in the binding affinity of domain III for NADP+ and for NADPH.

The binding of nucleotides to domain I proteins of the proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli as measured by equilibrium dialysis.

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherichia coli were overexpressed and purified. Nucleotide binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd = 32 microM and 120 microM, respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers, and at pH 7.5 in sodium phosphate buffer. NAD+ bound with lower affinity (Kd = 300 microM). NADPH bound only very weakly (Kd > 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M. & Hatefi, Y. (1993) J. Biol. Chem. 268. 17871-17877] found that mitochondrial transhydrogenase, and a proteolytically derived domain I fragment from that enzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-site reactivity for the interaction between the nucleotide ligand and the protein. However, our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I proteins using equilibrium dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monomeric units is evident. Reasons for the discrepancies between the work on bacterial and mitochondrial transhydrogenases are discussed.

Interaction of nucleotides with the NAD(H)-binding domain of the proton-translocating transhydrogenase of Rhodospirillum rubrum.

Transhydrogenase catalyzes the reduction of NADP+ by NADH coupled to the translocation of protons across a membrane. The polypeptide composition of the enzyme in Rhodospirillum rubrum is unique in that the NAD(H)-binding domain (called Ths) exists as a separate polypeptide. Ths was expressed in Escherichia coli and purified. The binding of nucleotide substrates and analogues to Ths was examined by one-dimensional proton nuclear magnetic resonance (NMR) spectroscopy and by measuring the quenching of fluorescence of its lone Trp residue. NADH and reduced acetylpyridine adenine dinucleotide bound tightly to Ths, whereas NAD+, oxidized acetylpyridine adenine dinucleotide, deamino-NADH, 5'-AMP and adenosine bound less tightly. Reduced nicotinamide mononucleotide, NADPH and 2'-AMP bound only very weakly to Ths. The difference in the binding affinity between NADH and NAD+ indicates that there may be an energy requirement for the transfer of reducing equivalents into this site in the complete enzyme under physiological conditions. Earlier results had revealed a mobile loop at the surface of Ths (Diggle, C., Cotton, N. P. J., Grimley, R. L., Quirk, P. G., Thomas, C. M., and Jackson, J. B. (1995) Eur. J. Biochem. 232, 315-326); the loop loses mobility when Ths binds nucleotide; the reaction involves two steps. This was more clearly evident, even for tight-binding nucleotides, when experiments were carried out at higher temperatures (37 degrees C), where the resonances of the mobile loop were substantially narrower. The binding of adenosine was sufficient to initiate loop closure; the presence of a reduced nicotinamide moiety in the dinucleotide apparently serves to tighten the binding. Two-dimensional 1H NMR spectroscopy of the Ths-5'-AMP complex revealed nuclear Overhauser effect interactions between protons of amino acid residues in the mobile loop (including those in a Tyr residue) and the nucleotide. This suggests that, in the complex, the loop has closed down to within 0.5 nm of the nucleotide.