Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli - Effects on catalytic and H+-pumping activities

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several tran smembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-t reated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-tre ated E. coli transhydrogenase. Despite harsh treatments with, e.g. detergents and denaturing agents, the a lpha and beta subunits remained tightly associated. A monoclonal antibody d irected towards the alpha subunit was strongly inhibitory, an effect that w as relieved by added rrI. In addition, rrI also reactivated the trypsin-dig ested E. coli enzyme in which domain I had been partly removed. This sugges ts that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II. Replacement of domain I of intact, as well as trypsin-digested, E. coli tra nshydrogenase with rrI resulted in a markedly different pH dependence of th e cyclic reduction of 3-acetyl-pyridine-NAD(+) by NADH in the presence of N ADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pum ping showed a less pronounced change in pH dependence, demonstrating the re gulatory role of domain II in these reactions.