Organization in the membrane of the N-terminal proton-translocating domain of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli.

The proton-translocating transmembrane pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta. The beta subunit has several membrane-spanning segments in the N-terminal region followed by a cytosolic C-terminal domain bearing a binding site for NADP(H). The N-terminal region contains at least one residue involved in the process of transmembrane proton translocation. Using site-directed mutagenesis cysteine residues were introduced at selected sites into the N-terminal region of the beta subunit. The pattern of labelling of these residues with 3-(N-maleimidyl propionyl)biocytin and other sulfhydryl reagents has shown that a model in which the N-terminal region of the beta subunit spans the membrane in eight segments is more likely than a previously proposed six segment model (Holmberg et al. (1994) Biochemistry 33, 7691-7700). The preferred model accounts for the site of labelling of a glutamate residue (Glu124) in the N-terminal domain by N,N-dicyclohexylcarbodiimide.