Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called “occluded” intermediary state of the reaction cycle of the intact enzyme. Despite a K_d in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP⁺ is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2′-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD⁺ by NADH, indicating that 3-acetylpyridine-NAD⁺ and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2′-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

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 The pH dependence of betaH91D, as well as betaH91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD(+) by NADPH, i.e., the reverse reaction, is optimal at a pH essentially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.     

Fast hydride transfer in proton-translocating transhydrogenase revealed in a rapid mixing continuous flow device

Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI(2).dIII(1) complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP(+) is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI(2).dIII(1) complexes catalyze reduction of NADP(+) by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s(-1), which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD(+)) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.     

The role of invariant amino acid residues at the hydride transfer site of proton-translocating transhydrogenase

Transhydrogenase couples proton translocation across a membrane to hydride transfer between NADH and NADP+. Previous x-ray structures of complexes of the nucleotide-binding components of transhydrogenase ("dI2dIII1" complexes) indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation. We have mutated three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase. In each mutant, turnover by the intact enzyme is strongly inhibited. Stopped-flow experiments using dI2dIII1 complexes show that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures of dI2dIII1 complexes carrying the mutations showed that their effects were restricted to the locality of the bound NAD(H). The results are consistent with the suggestion that in wild-type protein movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer.     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K(d) values) for NADPH (0.87 microM), NADP(+) (16 microM), NADH (50 microM), and NAD(+) (100-500 microM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K(d) values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K(d) values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidized.     

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.     

Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)(2)dIII(1). The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)(2)dIII(1) was <1% of that for the wild-type complex, and the deuterium isotope effect was significantly decreased. The nucleotide binding properties of the dI component in the complex were asymmetrically affected by the Gln-to-Asn mutation. In intact, membrane-bound transhydrogenase, the substitution completely abolished all catalytic activity. The results suggest that Gln132 in the wild-type enzyme behaves as a "tether" or a "tie" in the mutual positioning of the (dihydro)nicotinamide rings of NAD(H) and NADP(H) for hydride transfer during the conformational changes that are coupled to the translocation of protons across the membrane. This ensures that hydride transfer is properly gated and does not take place in the absence of proton translocation.     

Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same beta subunit has been demonstrated.     

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

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

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.     

A conformational change in the isolated NADP(H)-binding component (dIII) of transhydrogenase induced by low pH: a reflection of events during proton translocation by the complete enzyme?

Transhydrogenase couples the reduction of NADP(+) by NADH to inward proton translocation across the bacterial (or mitochondrial) membrane. Conformational changes in the NADP(H)-binding component of the enzyme (dIII) are central to the coupling mechanism. In the "open" state, NADP(H) bound to dIII can readily exchange with nucleotides in the solvent but hydride transfer [to/from NAD(H) bound to dI] is prevented. In the "occluded" state, bound NADP(H) cannot exchange with solvent nucleotides but the hydride transfer reaction is permitted. It was previously found that the conformational state of isolated, recombinant dIII is pH dependent. At neutral pH, the protein adopts a conformation resembling the occluded state, and at low pH, it adopts a conformation resembling the open state. The crystal structure of dIII indicates that the loop E "lid" might be largely responsible for the very high affinity of the protein for NADP(H). In this paper we show, using fluorescence resonance energy transfer, that the distance between the apex of loop E of isolated dIII, and the core of the protein, increases when the solution pH is lowered. This is consistent with the view that the lid is retracted to permit NADPH release during turnover of the complete enzyme.     

Protein-protein recognition, hydride transfer and proton pumping in the transhydrogenase complex

BACKGROUND: Membrane-bound ion pumps are involved in metabolic regulation, osmoregulation, cell signalling, nerve transmission and energy transduction. How the ion electrochemical gradient interacts with the scalar chemistry and how the catalytic machinery is gated to ensure high coupling efficiency are fundamental to the mechanism of action of such pumps. Transhydrogenase is a conformationally coupled proton pump linking a proton gradient to the redox reaction between NAD(H) and NADP(H). The enzyme has three components; dI binds NAD(H), dII spans the membrane and dIII binds NADP(H). RESULTS: The first crystal structure of a transhydrogenase dI component (from Rhodospirillum rubrum) has been determined at 2.0 A resolution. The monomer comprises two domains. Both are involved in dimer formation, and one has a Rossmann fold that binds NAD+ in a novel mode. The two domains can adopt different conformations. In the most closed conformation, the nicotinamide ring is expelled from the cleft between the two domains and is exposed on the outside of the protein. In this conformation it is possible to dock the structure of dI/NAD+ with that of a dIII/NADP+ complex to provide the first insights into the molecular basis of the hydride-transfer step. CONCLUSIONS: Analysis of the model of the dI/dIII complex identifies residues potentially involved in dI/dIII interaction and shows how domain motion in dI results in a shift in position of the nicotinamide ring of NAD+. We propose that this movement is responsible for switching between the forbidden and allowed states for hydride transfer during proton pumping.     

A change in ionization of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase regulates both hydride transfer and nucleotide release.

Transhydrogenase couples the transfer of hydride-ion equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the membrane. Coupling between transhydrogenation and proton translocation involves changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single-turnover burst of hydride transfer between bound nucleotides; subsequent turnover is limited by NADP(H) release. Stopped-flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site-directed mutagenesis. Two mutants with similar catalytic properties to those of the wild-type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affected by nucleotide occupancy of dIII was different in the two mutants, and hence two different procedures for determining the rate of nucleotide release were developed. The apparent first-order rate constants for NADP(+) release and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of the protonated and unprotonated forms of dIII are consistent with those expected of intermediates in the NADP(H)-binding-change mechanism. The ionisable group might be a component of the proton-translocation pathway in the complete enzyme.     

Structure and mechanism of proton-translocating transhydrogenase

Recent developments have led to advances in our understanding of the structure and mechanism of action of proton-translocating (or AB) transhydrogenase. There is (a) a high-resolution crystal structure, and an NMR structure, of the NADP(H)-binding component (dIII), (b) a homology-based model of the NAD(H)-binding component (dI) and (c) an emerging consensus on the position of the transmembrane helices (in dII). The crystal structure of dIII, in particular, provides new insights into the mechanism by which the energy released in proton translocation across the membrane is coupled to changes in the binding affinities of NADP(+) and NADPH that drive the chemical reaction.     

Active-site conformational changes associated with hydride transfer in proton-translocating transhydrogenase

Transhydrogenase couples the redox (hydride-transfer) reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The redox reaction is catalyzed at the interface between two components (dI and dIII) which protrude from the membrane. A complex formed from recombinant dI and dIII (the dI(2)dIII(1) complex) from Rhodospirillum rubrum transhydrogenase catalyzes fast single-turnover hydride transfer between bound nucleotides. In this report we describe three new crystal structures of the dI(2)dIII(1) complex in different nucleotide-bound forms. The structures reveal an asymmetry in nucleotide binding that complements results from solution studies and supports the notion that intact transhydrogenase functions by an alternating site mechanism. In one structure, the redox site is occupied by NADH (on dI) and NADPH (on dIII). The dihydronicotinamide rings take up positions which may approximate to the ground state for hydride transfer: the redox-active C4(N) atoms are separated by only 3.6 A, and the perceived reaction stereochemistry matches that observed experimentally. The NADH conformation is different in the two dI polypeptides of this form of the dI(2)dIII(1) complex. Comparisons between a number of X-ray structures show that a conformational change in the NADH is driven by relative movement of the two domains which comprise dI. It is suggested that an equivalent conformational change in the intact enzyme is important in gating the hydride-transfer reaction. The observed nucleotide conformational change in the dI(2)dIII(1) complex is accompanied by rearrangements in the orientation of local amino acid side chains which may be responsible for sealing the site from the solvent and polarizing hydride transfer.     

The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli

Proton-translocating nicotinamide nucleotide transhydrogenase is a conformationally driven pump which catalyzes the reversibel reduction of NADP(+) by NADH. Transhydrogenases contain three domains, i.e., the hydrophilic NAD(H)-binding domain I and the NADP(H)-binding domain III, and the hydrophobic domain II containing the proton channel. Domains I and III have been separately expressed and characterized structurally by, e.g. X-ray crystallography and NMR. These domains catalyze transhydrogenation in the absence of domain II. However, due to the absence of the latter domain, the reactions catalyzed by domains I and III differ significantly from those catalyzed by the intact enzyme. Mutagenesis of residues in domain II markedly affects the activity of the intact enzyme. In order to resolve the structure-function relationships of the intact enzyme, and the molecular mechanism of proton translocation, it is therefore essential to establish the structure and function of domain II and its interactions with domains I and III. This review describes some relevant recent results in this field of research.     

Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains

Transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound within soluble domains I and III, respectively, to proton translocation across membrane bound domain II. The cocrystal structure of Rhodospirillum rubrum TH domains I and III has been determined in the presence of limiting NADH, under conditions in which the subunits reach equilibrium during crystallization. The crystals contain three heterotrimeric complexes, dI(2)dIII, in the asymmetric unit. Multiple conformations of loops and side-chains, and NAD(H) cofactors, are observed in domain I pertaining to substrate/product exchange, and highlighting electrostatic interactions during the hydride transfer. Two interacting NAD(H)-NADPH pairs are observed where alternate conformations of the NAD(H) phosphodiester and conserved arginine side-chains are correlated. In addition, the stereochemistry of one NAD(H)-NADPH pair approaches that expected for nicotinamide hydride transfer reactions. The cocrystal structure exhibits non-crystallographic symmetry that implies another orientation for domain III, which could occur in dimeric TH. Superposition of the "closed" form of domain III (PDB 1PNO, chain A) onto the dI(2)dIII complex reveals a severe steric conflict of highly conserved loops in domains I and III. This overlap, and the overlap with a 2-fold related domain III, suggests that motions of loop D within domain III and of the entire domain are correlated during turnover. The results support the concept that proton pumping in TH is driven by the difference in binding affinity for oxidized and reduced nicotinamide cofactors, and in the absence of a difference in redox potential, must occur through conformational effects.     

Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core

The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.     

A hybrid of the transhydrogenases from Rhodospirillum rubrum and Mycobacterium tuberculosis catalyses rapid hydride transfer but not the complete, proton-translocating reaction

All transhydrogenases appear to have three components: dI, which binds NAD(H), and dIII, which binds NADP(H), protrude from the membrane, and dII spans the membrane. However, the polypeptide composition of the enzymes varies amongst species. The transhydrogenases of Mycobacterium tuberculosis and of Rhodospirillum rubrum have three polypeptides. Sequence analysis indicates that an ancestral three-polypeptide enzyme evolved into transhydrogenases with either two polypeptides (such as the Escherichia coli enzyme) or one polypeptide (such as the mitochondrial enzyme). The fusion steps in each case probably led to the development of an additional transmembrane helix. A hybrid transhydrogenase was constructed from the dI component of the M. tuberculosis enzyme and the dII and dIII components of the R. rubrum enzyme. The hybrid catalyses cyclic transhydrogenation but not the proton-translocating, reverse reaction. This shows that nucleotide-binding/release at the NAD(H) site, and hydride transfer, are fully functional but that events associated with NADP(H) binding/release are compromised. It is concluded that sequence mismatch in the hybrid prevents a conformational change between dI and dIII which is essential for the step accompanying proton translocation.