Role of Methionine-239, an Amino Acid Residue in the Mobile-Loop Region of the NADH-Binding Domain (Domain I) of Proton-Translocating Transhydrogenase

Abstract

Transhydrogenase couples the transfer of hydride equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The one-dimensional proton NMR spectrum of the recombinant NAD(H)-binding domain (domain I) of transhydrogenase from Rhodospirillum rubrum reveals well-defined resonances, several of which arise from a mobile loop at the 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 shifting of the mobile loop resonances consequent on NAD(H) binding indicate that the loop closes down on the protein surface. More NAD(H) had to be added to mutant domain I than to wild type to give comparable resonance broadening. The Kd of domain I for NADH, measured by equilibrium dialysis, was increased about three-fold by the Met239 mutations. Mutant and wild-type domain I were reconstituted with domain I-depleted membranes from R. rubrum, and with recombinant domain III of transhydrogenase. With membranes, the Km for acetylpyridine adenine dinucleotide during reverse transhydrogenation was 5x and > 6x greater in dI.M239I and dI.M239F, respectively, than in wild-type. Cyclic transhydrogenation (in membranes and the recombinant system) was substantially more inhibited (70% in dI.M239I, and 84% in dI.M239F) than either forward or reverse transhydrogenation. The docking affinities of dI.M239I and dI.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, and that in proteins with amino acid substitutions at this position, the binding affinity of NAD(H) is decreased, and the hydride transfer step is inhibited.

Interactions of the NADP(H)-Binding Domain III of Proton-Translocating Transhydrogenase fromEscherichiacoliwith NADP(H) and the NAD(H)-Binding Domain I Studied by NMR and Site-Directed Mutagenesis

“…The maximal rate of the reverse reaction catalyzed by mixtures of rrI and ecIII at pH 7.0 has been shown to be limited by the slow release of NADP + ( 7 , − . All cysteine mutants described in Table showed increased maximal reverse rates, which in most cases is explained by the decreased affinity for NADP(H) caused by the introduced mutation (cf.…”

“…Chemical shift mapping provides general information about the position and extent of the substrate-binding site surface, whereas activity measurements and determination of substrate content of mutant ecIII provides important information regarding the involvement of specific residues in substrate binding. The maximal rate of the reverse reaction catalyzed by the rrI‚ ecIII complex is limited by the release of NADP + (10)(11)(12)32). V max of the reverse reaction is thus another important indicator for how the NADP(H) affinity has changed due to a mutation of a certain amino acid residue.…”

“…Even in the absence of the transmembrane domain II, domain I and III from the same or different species form a catalytically active complex capable of catalyzing the various transhydrogenation reactions (6)(7)(8)10). However, the tight binding of NADP(H) and thus the limiting NADP(H) release results in low reaction rates for the reverse and forward reactions catalyzed by the dI‚dIII complex (7,(10)(11)(12)(13).…”

Structure and mechanism of proton‐translocating transhydrogenase

“…Upon NAD(H) binding, this ‘mobile loop’ closes down on the surface of the protein and makes contact with the nucleotide. Mutational analysis [65–68] shows that the loop is important in catalysis; it was suggested to have a role in the relative positioning of the nicotinamide rings of NADH and NADP + prior to hydride transfer.…”

The Alternating Site, Binding Change Mechanism for Proton Translocation by Transhydrogenase

“…Amino acid residues at the apex of the mobile loop approach those in the RQD loop, leading to partial enclosure of the bound NAD + . The feature was studied by NMR before emergence of the X-ray structures (29,(46)(47)(48). Because of its segmental mobility in the absence of nucleotides, resonances in the loop remain relatively unbroadened and are therefore easily detected.…”