The heterotrimer of the membrane-peripheral components of transhydrogenaseand the alternating-site mechanism of proton translocation

Transhydrogenase undergoes conformational changes to couple the redox react ion between NAD(H) and NADP(H) to proton translocation across a membrane. T he protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isother mal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recom binant dI and dIII from Rhodospirillum rubrum transhydrogenase readily form s a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence fo r the formation of a dI(2)dIII(2) tetramer using these techniques. The asym metry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complet e enzyme. The characteristics of nucleotide interaction with the isolated d l and dIII components and with the dI(2)dIII(1) heterotrimer were investiga ted. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affi nity of one of the two nucleotide-binding sites for NADH on the dI dimer wa s decreased about 17-fold in the dI(2)dIII(1), complex; the other binding s ite was unaffected. These observations lend strong support to the alternati ng-site mechanism.