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 beta His91 and beta Asn222. The pH dependence of beta H91D, as well as beta T91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD(+) by NADPH, i.e., the reverse reaction, is optimal at a pH essentially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 beta 91 position. It is therefore concluded that the wild-type transhydrogenase is regulated by the degree of protonation of beta His91. The mechanisms of the interactions between dI+dIII and dII are suggested to involve pronounced conformational changes in a 'hinge' region around beta R265. (C) 2000 Elsevier Science B.V. All rights reserved.