The heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation.

Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.

The crystal structure of an asymmetric complex of the two nucleotide binding components of proton-translocating transhydrogenase.

BACKGROUND: Membrane-bound ion translocators have important functions in biology, but their mechanisms of action are often poorly understood. Transhydrogenase, found in animal mitochondria and bacteria, links the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Linkage is achieved through changes in protein conformation at the nucleotide binding sites. The redox reaction takes place between two protein components located on the membrane surface: dI, which binds NAD(H), and dIII, which binds NADP(H). A third component, dII, provides a proton channel through the membrane. Intact membrane-located transhydrogenase is probably a dimer (two copies each of dI, dII, and dIII). RESULTS: We have solved the high-resolution crystal structure of a dI:dIII complex of transhydrogenase from Rhodospirillum rubrum-the first from a transhydrogenase of any species. It is a heterotrimer, having two polypeptides of dI and one of dIII. The dI polypeptides fold into a dimer. The loop on dIII, which binds the nicotinamide ring of NADP(H), is inserted into the NAD(H) binding cleft of one of the dI polypeptides. The cleft of the other dI is not occupied by a corresponding dIII component. CONCLUSIONS: The redox step in the transhydrogenase reaction is readily visualized; the NC4 atoms of the nicotinamide rings of the bound nucleotides are brought together to facilitate direct hydride transfer with A-B stereochemistry. The asymmetry of the dI:dIII complex suggests that in the intact enzyme there is an alternation of conformation at the catalytic sites associated with changes in nucleotide binding during proton translocation.

Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum.

Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria. The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods. This is the first description of the structure of dIII from a bacterial source. The protein adopts a Rossmann fold: an open, twisted, parallel beta-sheet, flanked by helices. However, the binding of NADP(+) to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first betaalphabetaalphabeta motif, and the nicotinamide with the second. Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified. The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.

Crystallization of the dI component of transhydrogenase, a proton-translocating membrane protein.

Nicotinamide nucleotide transhydrogenase couples the exchange of a hydride-ion equivalent between NAD(H) and NADP(H) to the translocation of protons across an energy-transducing membrane. Peripheral components of 380 and 200 residues bind NAD(H) (dI) and NADP(H) (dIII), respectively, while a third component forms a membrane-spanning region (dII). The NAD(H)-binding component dI of Rhodospirillum rubrum transhydrogenase has been crystallized in a form which diffracts to beyond 3.0 A resolution and is in space group P2 or P2(1), with unit-cell parameters a = 69.3, b = 117.8, c = 106.6 A, beta = 107.2 degrees and two dimers in the asymmetric unit. The sequence of the dI component is similar to that of alanine dehydrogenase. A full structure determination will lead to important information on the mode of action of this proton pump and will permit the comparison of the structure-function relationships of dI with those of alanine dehydrogenase.

The high-resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human heart mitochondria.

BACKGROUND: Transhydrogenase, located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping. The protein comprises three subunits termed dI, dII and dIII. The dII component spans the membrane. The dI component, which contains the binding site for NAD(+)/NADH, and the dIII component, which has the binding site for NADP(+)/NADPH, protrude from the membrane. Proton pumping is probably coupled to changes in the binding affinities of dIII for NADP(+) and NADPH. RESULTS: The first X-ray structure of the NADP(H)-binding component, dIII, of human heart transhydrogenase is described here at 2.0 A resolution. It comprises a single domain resembling the classical Rossmann fold, but NADP(+) binds to dIII with a reversed orientation. The first betaalphabetaalphabeta motif of dIII contains a Gly-X-Gly-X-X-Ala/Val 'fingerprint', but it has a different function to that in the classical Rossmann structure. The nicotinamide ring of NADP(+) is located on a ridge where it is exposed to interaction with NADH on the dI subunit. Two distinctive features of the dIII structure are helix D/loop D, which projects from the beta sheet, and loop E, which forms a 'lid' over the bound NADP(+). CONCLUSIONS: Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dII. Changes in ionisation and conformation in helix D/loop D, resulting from proton translocation through dII, are thought to be responsible for the changes in affinity of dIII for NADP(+) and NADPH that drive the reaction.

Evidence for the stabilization of NADPH relative to NADP(+) on the dIII components of proton-translocating transhydrogenases from Homo sapiens and from Rhodospirillum rubrum by measurement of tryptophan fluorescence.

A unique Trp residue in the recombinant dIII component of transhydrogenase from human heart mitochondria (hsdIII), and an equivalent Trp engineered into the dIII component of Rhodospirillum rubrum transhydrogenase (rrdIII.D155W), are more fluorescent when NADP(+) is bound to the proteins, than when NADPH is bound. We have used this to determine the occupancy of the binding site during transhydrogenation reactions catalysed by mixtures of recombinant dI from the R. rubrum enzyme and either hsdIII or rrdIII.D155W. The standard redox potential of NADP(+)/NADPH bound to the dIII proteins is some 60-70 mV higher than that in free solution. This results in favoured reduction of NADP(+) by NADH at the catalytic site, and supports the view that changes in affinity at the nucleotide-binding site of dIII are central to the mechanism by which transhydrogenase is coupled to proton translocation across the membrane.

Structural changes in the recombinant, NADP(H)-binding component of proton translocating transhydrogenase revealed by NMR spectroscopy.

We have analysed 1H, 15N-HSQC spectra of the recombinant, NADP(H)-binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein. Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein. These observations reflect the structural changes resulting from hydride transfer. The interactions between the recombinant, NADP(H)-binding component and its partner, NAD(H)-binding protein, are complicated. Helix B of the recombinant, NADP(H)-binding component may play an important role in the binding process.

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.

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.

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.

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.

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.

Mutation of Tyr235 in the NAD(H)-binding subunit of the proton-translocating nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum affects the conformational dynamics of a mobile loop and lowers the catalytic activity of the enzyme.

The Tyr residue in the mobile loop region of the soluble, domain I polypeptide (called Ths) of the proton-translocating transhydrogenase from Rhodospirillum rubrum has been substituted by Asn and by Phe. The recombinant proteins were expressed at high levels in Escherichia coli and purified to homogeneity. The two well defined resonances at 6.82 and 7.12ppm, observed in the one-dimensional proton NMR spectrum of wild-type protein, and previously attributed to the Tyr residue, were absent in both mutants. In the Tyr235 --> Phe mutant Ths, they were replaced by two new resonances at 7.26 and 7.33 ppm, characteristic of a Phe residue. In both mutants, narrow resonances attributable to Met residues (and in the Tyr235 --> Phe mutant, resonances attributable to Ala residues) were shifted relative to the wild type, but other features in the NMR spectra were unaffected. The conformational dynamics of the mobile loop closure in response to nucleotide binding by the protein were altered in the two mutants. The fluorescence emission from Trp72 was unaffected by both Tyr substitutions, and the fluorescence was still quenched by NADH. The mutant Ths proteins bound to chromatophore membranes depleted of their native Ths with undiminished affinity. In these reconstituted systems, the Km values for thio-NADP+ and NADH, during light-driven transhydrogenation, were similar to those of wild-type, but the kcat values were decreased about 2-fold. In reverse transhydrogenation, the Kmvalues for NADPH were slightly decreased in the mutants relative to wild-type, but those for acetyl pyridine adenine dinucleotide were increased about 10- and 13-fold, respectively, and the kcat values were decreased about 2- and 5-fold, respectively, in the Tyr235 --> Phe and Tyr235 --> Asn mutants. It is concluded that Tyr235 may contribute to the process of nucleotide binding and that substitution of this residue prevents proper functioning of the mobile loop in catalysis.

Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli.

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

The involvement of NADP(H) binding and release in energy transduction by proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli.

Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli. Consistent with recent evidence [Hutton, M., Day, J., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051], at low pH and salt concentration, the enzyme catalysed rapid reduction of the NAD+ analogue AcPdAD+ by a combination of NADH and NADPH. At saturating concentrations of NADPH, the dependence of the steady-state rate on the concentrations of NADH and AcPdAD+ indicated that, with respect to these two nucleotides, the reaction proceeds by a ping-pong mechanism. High concentrations of either NADH or AcPdAD+ led to substrate inhibition. These observations support the view that, in this reaction, NADP(H) remains bound to the enzyme: AcPdAD+ is reduced by enzyme-bound NADPH, and NADH is oxidised by enzyme-bound NADP+, in a cyclic process. When this reaction was carried out with [4A-2H]NADH replacing [4A-1H]NADH, the rate was decreased by 46%, suggesting that the H- transfer steps are rate-limiting. In simple 'reverse' transhydrogenation, the reduction of AcPdAD+ was slower with [4B-2H]NADPH than with [4B-1H]NADPH when the reaction was performed at pH 8.0, but there was no deuterium isotope effect at pH 6.0. This indicates that H- transfer is rate-limiting at pH 8.0 and supports our earlier suggestion that NADP+ release from the enzyme is rate-limiting at low pH. The lack of a deuterium isotope effect in the reduction of thio-NADP+ by NADH at low pH is also consistent with the view that NADPH release from the enzyme is slow under these conditions. A steady-state rate equation is derived for the reduction of AcPdAD+ by NADPH plus NADH, assuming operation of the cyclic pathway. It adequately accounts for the pH dependence of the enzyme, for the features described above and for kinetic characteristics of E. coli transhydrogenase described in the literature.

Cloning and sequencing of the genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and the implications for the domain structure of the enzyme.

The genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated pntAA, pntAB and pntB, whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from Escherichia coli (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from R. rubrum, equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the alpha polypeptide of E. coli transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the alpha polypeptide of the E. coli transhydrogenase. PntB corresponds to the E. coli beta polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the E. coli and bovine mitochondrial enzymes. The two tryptic fragments from the native R. rubrum soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.