The binding of nucleotides to domain I proteins of the proton-translocating transhydrogenases from <em>Rhodospirillum rubrum</em> and <em>Escherichia coli</em> as measured by equilibrium dialysis.

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherchia coli were overexpressed and purified. Nucleoude binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (K_d = 32 μM and 120 μM). respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers and at pH 7.5 in sodium phosphate buffer, NAD⁺hound with lower affinity (K_d = 300 μM). NADPH bound only very weakly (K_d1> 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M & Hatefi, Y. (1993) J. Biol. Chem, 268, 1781-17877 ] found that mitochondrial transhydrogenase, and a proteolytically derrived domain I fragment from that exnzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-sites reactivity for the interaction between the nucleotide ligand and the protein. However our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I protein using equilibrum dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monochondrial transhydrogenases are discussed. [ABSTRACT FROM AUTHOR]