The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides

In its forward direction, transhydrogenase couples the reduction of NADP+ by NADH to the outward translocation of protons across the membrane of bacteria and animal mitochondria. The enzyme has three components: dI and dIII protrude from the membrane and dII spans the membrane. Hydride transfer takes place between nucleotides bound to dI and dIII. Studies on the kinetics of a lag phase at the onset of a “cyclic reaction” catalysed by complexes of the dI and dIII components of transhydrogenase from Rhodospirillum rubrum, and on the kinetics of fluorescence changes associated with nucleotide binding, reveal two features. Firstly, the binding of NADP+ and NADPH to dIII is extremely slow, and is probably limited by the conversion of the occluded to the open state of the complex. Secondly, dIII can also bind NAD+ and NADH. Extrapolating to the intact enzyme this binding to the “wrong” site could lead to slip: proton translocation without change in the nucleotide redox state, which would have important consequences for bacterial and mitochondrial metabolism.

Research Highlights
► Transhydrogenase is utilised for NADP+ reduction in bacteria and mitochodria.
► Subcomplexes of transhydrogenase bind NADP+ and NADPH very slowly.
► Nucleotide binding is restricted by conversion of the occluded to the open state.
► Unexpectedly, NAD+ and NADH can bind into the NADP(H)-binding site.
► The metabolic significance of “wrong”-site binding is discussed.