MUTATIONS AT TYROSINE-235 IN THE MOBILE LOOP REGION OF DOMAIN-I PROTEIN OF TRANSHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM STRONGLY INHIBIT HYDRIDE TRANSFER

Transhydrogenase from mitochondrial and bacterial membranes couples pr oton translocation to hydride transfer between NAD(H) and NADP(H). The enzyme has three domains, of which domains I and III protrude from th e membrane. These possess the NAD(H)- and NADP(H)-binding sites, respe ctively, whereas domain II spans the membrane. In domain I there is a mobile loop which emanates from the surface of the protein, but which closes down upon NAD(H) binding. In this report we show that the NADP( H)-dependent reduction of acetylpyridine adenine dinucleotide by NADH catalysed by Rhodospirillum rubrum transhydrogenase has 'ping-pong' ki netics, confirming that the reaction is cyclic. We then describe the k inetic and thermodynamic properties of mutants of recombinant domain I protein from the R. rubrum enzyme, in which Tyr-235 in the mobile loo p has been substituted with Phe or Asn residues (dI.Y235F and dI.Y235N , respectively). (1) Equilibrium dialysis measurements show that dI.Y2 35F and dI.Y235N bind NADH more weakly than wild-type domain I protein (the K-d increases twofold and fourfold, respectively). (2) Reverse t ranshydrogenation rates (in steady state) of domain I-depleted membran e vesicles reconstituted with either dI.Y235F or dI.Y235N are inhibite d by about 50% and 78%, respectively, relative to those obtained in re constitutions with wild-type domain I protein. (3) Reverse transhydrog enation rates (in steady state) of mixtures of recombinant domain III protein and either dI.Y235F or dI.Y235N are inhibited only by about 10 % and 20%, respectively, relative to those obtained in mixtures with w ild-type protein. (4) Forward transhydrogenation rates (in both the co mplete enzyme and in domain I:III complexes) are inhibited even less b y the mutations than the reverse reactions, (5) In contrast with (1), (2) and (3), cyclic transhydrogenation was strongly inhibited in both the reconstituted membrane system and in the recombinant domain I:III complexes (only 7-8% activity remains with dI.Y235F, and only 2-3% wit h dI.Y235N). It was recently established that, in contrast to forward and reverse transhydrogenation, the cyclic reaction is substantially l imited by the rate of hydride transfer. It is therefore concluded that mutations at Tyr-235 in the mobile loop severely disrupt the hydride transfer step in the catalytic reaction of transhydrogenase.