Transhydrogenase comprises three domains. Domains I and I11 are peripheral to the membrane and possess the NAD(H)-and NADP(H)-binding sites, respectively, and domain 11 spans the membrane. Domain TIT 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 111 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 Ill were 0.03 SKI and S.6X10-4s-1, respectively. In the absence of domain I1 a mixture of the recombinant domain 111 protein, plus the previously described recombinant domain 1 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 111. Similarly, the mixture catalysed reduction of thio-NADP' by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain 111. 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 111 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 111 proteins during the time taken for a single domain I11 to release its NADP ' , whereas (b) the cyclic reaction is rapid on the timescale of formation and breakdown of the domain I . 111 complex. The rate of the hydride transfer reaction was similar in thc domain I I I11 complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I . 111 complex due to the greatly decreased rates of release of NADP' and NADPH. It is concluded that, in the complete enzyme, conforniational changes in the membrane-spanning domain 11, which result from proton translocation, lead to changes in the binding affinity of domain 111 for NADP' and for NADPH.

The mechanism, by which transhydrogenase couples transfer of H-equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichiu coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP' or NADPH. This indicates that the rates of release of NADP' and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP' and NADPH are accompanied by the release of H' from the enzyme and (b) increased ionic strength decreases the value of the pK, of the group responsible for H' release. Modification of the enzyme with N,Wdicyclohexylcarbodiimide led to inhibition of the rate of release of NADP' and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD', NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates.It is considered that the binding and release of H' , which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state.

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 ϩ /NADH, and domain III for NADP ϩ /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 ϩ analogue, acetylpyridine adenine dinucleotide (AcPdAD ϩ ). 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 ϩ (kϷ0.03 s Ϫ1 ). Phase A of the burst (kϷ600 s Ϫ1 ) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (kϷ10Ϫ50 s Ϫ1 ), 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 Keywords : transhydrogenase; stopped flow; proton translocation; recombinant protein; membrane protein.Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across a membrane (for reviews,(1) The enzyme is found in animal mitochondria and in bacteria. Probably, under most physiological conditions, the reaction is driven to the right, towards NADPH formation, by the proton motive force generated by the respiratory (or photosynthetic) electron-transport chain.Transhydrogenase has three domains. Domains I and III protrude from the membrane and possess the nucleotide-binding sites ; domain I for NAD ϩ and NADH, and domain III for NADP ϩ and NADPH [1Ϫ3]. Domain II spans the membrane and might comprise 10Ϫ12 transmembrane helices [4].Recombinant forms of domain I from Rhodospirillum rubrum [5,6] and Escherichia coli [7,8] and purified. The isolated domains bind their respective nucleotides with high specificity and affinity [5Ϫ10, 11]. A mixture of the two domains from the R. rubrum protein catalyses transhydrogenation, even in the absence of the membrane-spanning domain II [6,9], but the rates of the 'forward' and 'reverse' transhydrogenation reactions catalysed by the mixture are very slow Ϫ they are profoundly limited by release of product NADPH (k off Ϸ 5ϫ10 Ϫ4 s Ϫ1 ) and NADP ϩ (k off Ϸ 0.03 s Ϫ1 ), respectively, from domain III [9]. Evidently, in the complete enzyme, release of NADP(H) is accelerated by domain II. The mixture of domains I and III...

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.Transhydrogenase is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria. It couples the transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across the membrane. The net reaction is as follows.For many years, the question as to whether hydride transfer between the nucleotides is direct or indirect has been a matter of controversy. It is central to our understanding of the energycoupling reactions. Transhydrogenase comprises three domains. Domains I and III protrude from the membrane (on the matrix side in mitochondria and on the cytoplasmic side in bacteria). Domain II spans the membrane. There are separate sites on the enzyme for NAD(H) and for NADP(H); the former is located on domain I, and the latter on domain III (for reviews, see Refs. 1-3).The results of some early experiments on transhydrogenases from mitochondria and from Rhodospirillum rubrum were interpreted as evidence for the existence of a stable, reducedenzyme intermediate (4 -6). It was implied that a functional group on the enzyme, presumably either an amino acid residue or an unidentified prosthetic group, can serve alternately as a hydride acceptor and hydride donor. For example,where E(H) represents the reduced-enzyme intermediate. However, in subsequent work other plausible explanations were found for the earlier data (7, 8). Moreover, the conclusions from steady-state kinetic analysis of transhydrogenase from various sources (7, 9 -11) have been interpreted as evidence that the reaction proceeds through the formation of a ternary complex of enzyme and nucleotide substrates. The addition of nucleotides is random, and fast, relative to the rate of a subsequent step in turnover. The reaction does not appear to take place via a substituted enzyme mechanism, and therefore the existence of a reduced enzyme intermediate, which is stable in the absence of nucleotide, is unlikely (viz. reactions exemplified by Equations 2 and 3). However, the steady-state data do not rule out the possible existence of a reduced enzyme intermediate within the ternary complex, that is a reaction of the following type.The possibility that Cys residues in the polypeptide chain might serve as reducible intermediates in hydride transfer (see, for example, Refs. 1 and 12) has been eliminated by amino acid sequence comparisons, there are no conserved Cys residues in transhydrogenases from different species, and by the fact that complete Cys replacement has only a minimal effect on transhydrogenation activity (13). It is unlikely that other amino acid residues have redox potentials in the appropriate range to serve as intermediates in transhydrogenation between NAD(H) and NADP(H). It ha...

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).