Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E-coli transhydrogenase

The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha -helices, and is responsible for proton transport. In the present investigation the segment beta C260-beta S266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for beta S266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the beta C260-beta R266 region revealed an increased accessibility in the presence of NADP(H) bound to domain IH; an opposite effect was observed for beta S266. A beta 213-beta R265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The beta S260C, beta K261C, beta A262C, beta M263, and beta N264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several beta R265 mutants and the beta 213C mutant showed inhibited proton-coupled reactions but also markedly increased K-m(NADPH) values. It is concluded that the mobile hinge region beta C260-beta S266 and the beta D213- beta R265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.