Interactions between Transhydrogenase and Thio-nicotinamide Analogues of NAD(H) and NADP(H) Underline the Importance of Nucleotide Conformational Changes in Coupling to Proton Translocation

Abstract

Transhydrogenase couples the reduction of NADP؉ by NADH to inward proton translocation across mitochondrial and bacterial membranes. The coupling reactions occur within the protein by long distance conformational changes. In intact transhydrogenase and in complexes formed from the isolated, nucleotide-binding components, thio-NADP(H) is a good analogue for NADP(H), but thio-NAD(H) is a poor analogue for NAD(H). Crystal structures of the nucleotide-binding components show that the twists of the 3-carbothiamide groups of thio-NADP ؉ and of thio-NAD ؉ (relative to the planes of the pyridine rings), which are defined by the dihedral, X am , are altered relative to the twists of the 3-carboxamide groups of the physiological nucleotides. The finding that thio-NADP ؉ is a good substrate despite an increased X am value shows that approach of the NADH prior to hydride transfer is not obstructed by the S atom in the analogue. That thio-NAD(H) is a poor substrate appears to be the result of failure in the conformational change that establishes the ground state for hydride transfer. This might be a consequence of restricted rotation of the 3-carbothiamide group during the conformational change.Transhydrogenase is found in the inner membrane of animal mitochondria and in the cytoplasmic membrane of bacteria. The enzyme provides NADPH for biosynthesis and for reduction of glutathione, and in some mammalian tissues, it probably participates in the regulation of flux through the tricarboxylic acid cycle (1, 2). Under most physiological conditions transhydrogenase is driven in the "forward" direction by the proton electrochemical gradient (⌬p) generated by respiratory (or photosynthetic) electron transport.There is general agreement that coupling between the redox reaction and proton translocation is mediated by changes in protein conformation, although the character of these conformational changes is not known (reviewed in Refs. 3-5). Coupling mechanisms that involve large conformational changes operating over considerable distances are emerging as a common feature in proteins that translocate solutes/ions across membranes, and the amenable properties of transhydrogenase make it an attractive model in the search for fundamental principles. The enzyme has three components. The dI component, which binds NAD ϩ and NADH, and the dIII component, which binds NADP ϩ and NADPH, are extrinsic proteins protruding from the membrane (on the matrix side in mitochondria and on the cytoplasmic side in bacteria), and dII spans the membrane. The enzyme is essentially a "dimer" of two dI-dIIdIII "monomers," although the polypeptide composition is variable among species. Crystal structures of Rhodospirillum rubrum dI (6, 7), bovine dIII (8), human dIII (9, 10), and R. rubrum dI 2 dIII 1 complex (11,12), and an NMR structure of R. rubrum dIII (13) have recently been published. Studies on the transient state kinetics of transhydrogenation reveal that the redox reaction between the two nucleotides is direct (14,15). Thus, the nicotinamide and dihydr...

Transhydrogenation Reactions Catalyzed by Mitochondrial NADH−Ubiquinone Oxidoreductase (Complex I)

“…Within the active site, steric interactions may hinder the stacking of the nicotinamide and isoalloxazine rings required for hydride transfer. In transhydrogenase, subtle effects on the conformational changes which produce the ground state for hydride transfer have recently been revealed in studies of thionicotinamide nucleotide analogues (48). For NADPH, the additional phosphate may disrupt interactions between the adenine rings and aromatic residues at the entrance to the NADH-binding cavity (36), preventing the nicotinamide headgroup attaining the correct position.…”

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

“…This single atom substitution relative to NADP ϩ can result in altered binding characteristics in nucleotide-binding proteins (51,52,66), and it may also cause larger absorbance shifts near 510 nm (50, 67). 4 We encountered some difficulty with excessive equilibration/stabilization times for the FMN ox/sq couple during oxidative titrations of eNOSr.…”

The mechanism of discrimination between oxidized and reduced coenzyme in the aldehyde dehydrogenase domain of Aldh1l1

“…It is likely that the bulky sulfur atom of thio-NADP + substituting for the carboxamide oxygen of NADP + cannot be accommodated in the extended conformation, and its repulsion from the carboxyl group of the glutamate results in the nicotinamide moiety being expelled from the active site. Of note, thio-NADP + appears in the extended conformation in the structures of DHFR [32] and transhydrogenase [33], the fact suggesting that this conformation is possible. Thus, we suggest that thio-NADP + bound to C t -FDH represents the conformation of the natural coenzyme on its way out after the completion of the hydride transfer.…”

“…It should be noted that Glu673 is also likely to participate in the first stage of coenzyme discharging, through energetically unfavorable interactions with carboxamide of nicotinamide, thus cooperating with Cys707 in dissociation of the nicotinamide from the catalytic center. In this regard, the glutamate could discriminate between different orientations of the planes of the carboxamide group relative to the nicotinamide ring in the oxidized and reduced coenzyme, a mechanism described for other enzymes [32, 33]. …”