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 proton translocation to hydride transfer between NAD(H) and NADP(H). The enzyme has three domains, of which domains I and III protrude from the membrane. These possess the NAD(H)- and NADP(H)-binding sites, respectively, 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' kinetics, confirming that the reaction is cyclic. We then describe the kinetic and thermodynamic properties of mutants of recombinant domain I protein from the R. rubrum enzyme, in which Tyr-235 in the mobile loop has been substituted with Phe or Asn residues (dI.Y235F and dI.Y235N, respectively). (1) Equilibrium dialysis measurements show that dI.Y235F and dI.Y235N bind NADH more weakly than wild-type domain I protein (the Kd increases twofold and fourfold, respectively). (2) Reverse transhydrogenation rates (in steady state) of domain I-depleted membrane vesicles reconstituted with either dI.Y235F or dI.Y235N are inhibited by about 50% and 78%, respectively, relative to those obtained in reconstitutions with wild-type domain I protein. (3) Reverse transhydrogenation 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 wild-type protein. (4) Forward transhydrogenation rates (in both the complete enzyme and in domain I:III complexes) are inhibited even less by 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% with dI.Y235N). It was recently established that, in contrast to forward and reverse transhydrogenation, the cyclic reaction is substantially limited 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.
Bizouarn
T
T
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Grimley
R
R
Diggle
C
C
Thomas
C M
CM
Jackson
J B
JB
D006859:Hydrogen / Q000378:metabolism*; D009154:Mutation; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D014443:Tyrosine / Q000737:chemistry
D011994:Recombinant Proteins; D009243:NAD; D014443:Tyrosine; D009249:NADP; D006859:Hydrogen; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases