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.

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.

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.z 1999 Federation of European Biochemical Societies.

Transhydrogenase, which is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria, catalyzes the reaction shown below.A single proton is translocated across the membrane, from the p-aqueous phase (the "outside" of intact mitochondria and bacteria) to the n-aqueous phase (the "inside"), for each hydride equivalent transferred from NADH to NADP ϩ . Under most conditions, this is the in vivo direction; the reaction is driven by the proton electrochemical gradient (⌬p) resulting from the action of respiratory (or photosynthetic) electron transport. For recent reviews, see Refs. 1-3.The gross structure of transhydrogenases from different species is strikingly invariant. The enzyme has three components; dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude from the membrane (on the cytoplasmic side in bacteria, and the matrix side in mitochondria), and dII spans the membrane. Crystal structures of the dIII components of human and bovine transhydrogenases were recently described (4 -6), and the solution structure of the Rhodospirillum rubrum equivalent was determined by NMR.1 The basic fold of dIII is similar to the classical, dinucleotide-binding domain of lactate dehydrogenase, but NADP ϩ is bound with an unusual, "reversed" orientation. The nicotinamide ring of the bound NADP ϩ is exposed on a ridge of dIII. A homology model of dI (8), based on the crystal structure of the sequence-related alanine dehydrogenase (9), suggests that the nicotinamide ring of NADH is located in a deep cleft. It was proposed that, in the complete transhydrogenase, the ridge of dIII inserts into the cleft of dI to bring the nicotinamide rings of the two nucleotides into apposition to effect direct hydride transfer (5, 6). The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).Kinetic studies of transhydrogenase have also developed, following discoveries that fragments of the protein retain their nucleotide binding and some catalytic capacity. Thus, recombinant dI and dIII proteins, which bind their cognate nucleotides, have now been isolated from a number of species (11-16); mixtures of these proteins, even from different species, catalyze transhydrogenation reactions, albeit with properties that are modified relative to those of the complete enzyme. Transient state experiments, in particular, have revealed useful information on the hydride transfer step (17)(18)(19). Without exception, experiments with dI⅐dIII complexes have been carried out with nucleotide analogues having altered absorbance spectra to facilitate measurement of the rate of reaction. In this report we describe transient state experiments with the physiological nucleotides. A procedure is described, in which singl...

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.