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 loca ted on the cytosolic side of the membrane, whereas the hydrophobic domain I I is composed of 13 transmembrane alpha -helices, and is responsible for pr oton transport. In the present investigation the segment beta C260-beta S26 6 connecting domain II and III was characterized primarily because of its a ssumed 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, bindi ng studies of the fluorescent maleimide derivative MIANS to each cysteine i n 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 fo r 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 S 260C, beta K261C, beta A262C, beta M263, and beta N264 mutants showed a pro nounced 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 mo bile hinge region beta C260-beta S266 and the beta D213- beta R265 salt bri dge play a crucial role in the communication between the proton translocati on/binding events in domain II and binding/release of NADP(H) in domain III .