The transmembrane domain and the proton channel in proton-pumping transhydrogenases

Proton-pumping nicotinamide nucleotide transhydrogenases are composed of th ree main domains, the NAD(H)-binding and NADP(H)-binding hydrophilic domain s I (dI) and III (dIII), respectively, and the hydrophobic domain II (dII) containing the assumed proton channel, dII in the Escherichia coli enzyme h as 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 His 91 and beta Asn222. The pH dependence of beta H91D, as well as beta T91C (u npublished), when compared to that of wild type shows that reduction of 3-a cetylpyridine-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 posi tion. It is therefore concluded that the wild-type transhydrogenase is regu lated by the degree of protonation of beta His91. The mechanisms of the int eractions between dI+dIII and dII are suggested to involve pronounced confo rmational changes in a 'hinge' region around beta R265. (C) 2000 Elsevier S cience B.V. All rights reserved.