Biochimica et Biophysica Acta 1997-11-10

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.

T Bizouarn, S Stilwell, J Venning, N P Cotton, J B Jackson

Index: Biochim. Biophys. Acta 1322(1) , 19-32, (1997)

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Abstract

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.