ROLE OF METHIONINE-239, AN AMINO-ACID RESIDUE IN THE MOBILE-LOOP REGION OF THE NADH-BINDING DOMAIN (DOMAIN-I) OF PROTON-TRANSLOCATING TRANSHYDROGENASE

Transhydrogenase couples the transfer of hydride equivalents between N AD(H) and NADP(H) to proton translocation across a membrane, The one-d imensional proton NMR spectrum of the recombinant NAD(H)-binding domai n (domain I) of transhydrogenase from Rhodospirillum rubrum reveals we ll-defined resonances, several of which arise from a mobile loop at th e protein surface. Four have been assigned to Met residues (MetA-MetD) . Substitution of Met239 with either Ile (dI.M239I) or Phe (dI.M239F) resulted in loss of MetA from the NMR spectrum, Broadening and shiftin g of the mobile loop resonances consequent on NAD(H) binding indicate that the loop closes down on the protein surface, More NAD(II) had to be added to mutant domain I than to wild type to give comparable reson ance broadening, The Kd of domain I for NADH, measured by equilibrium dialysis, was increased about three-fold by the Met239 mutations. Muta nt and wild-type domain I were reconstituted with domain I-depleted me mbranes from R. rubrum, and with recombinant domain III of transhydrog enase. With membranes, the K-m for acetylpyridine adenine dinucleotide during reverse transhydrogenation Was 5 x and >6 x greater in dI.M239 I and dI.M239F, respectively, than in wild-type. Cyclic transhydrogena tion (in membranes and the recombinant system) was substantially more inhibited (70% in dI.M239I, and 84% in dI.M239F) than either forward o r reverse transhydrogenation, The docking affinities of dI.M239I and d I.M239F to the depleted membranes were similar to those of wild-type, It is concluded that Met239 is MetA in the mobile loop of domain I, an d that in proteins with amino acid substitutions at this position, the binding affinity of NAD(H) is decreased, and the hydride transfer ste p is inhibited.