Catalytic properties of hybrid complexes of the NAD(H)-binding and NADP(H)-binding domains of the proton-translocating transhydrogenases from Escherichia coli and Rhodospirillum rubrum

Transhydrogenase couples reversible hydride transfer from NADH to NADP(+) t o proton translocation across the inner membrane in mitochondria and the cy toplasmic membrane in bacteria. The enzyme is composed of three parts. Doma in I (dI) and domain III (dIII) are water soluble and contain the binding s ites for NAD(H) and NADP(H), respectively; domain II (dII) spans the membra ne. In the present investigation, dI from Rhodospirillum rubrum (rrI) and E scherichia coli (ecI), and dIII from R. rubrum (rrIII) and E, coli (ecIII) were overexpressed in E. coli and subsequently purified. Also, a preparatio n of a partially degraded E. coli transhydrogenase (ec beta) was examined. Catalytic activities were analyzed in various dI+dIII and dI+ec beta combin ations. The abilities of the different dI+dIII combinations to catalyze cyc lic transhydrogenation, i.e., the reduction of AcPyAD(+) by NADH mediated v ia tightly bound NADP(H) in dIII, varied in the order: rrI+ecIII approximat e to rrI+rrIII > rrI+ec beta >> ecI+ecIII; no measurable activities for ecI +rrIII and ecI+ec beta were detected. Thus, rrI has a much greater apparent affinity than ecI for ecIII or rrIII or ec beta. The pH dependences of the cyclic reaction seem to be determined by scalar protonation events on dI, both in rrI+rrIII and ecI+ecIII mixtures as well as in the wild-type R. rub rum and possibly in the E. coli enzyme. Higher reverse activities for rrI+e c beta than for rrI+ecIII confirmed the regulatory role of dII for the asso ciation and dissociation rates of NADP(H).