THE BINDING OF NUCLEOTIDES TO DOMAIN-I PROTEINS OF THE PROTON-TRANSLOCATING TRANSHYDROGENASES FROM RHODOSPIRILLUM-RUBRUM AND ESCHERICHIA-COLI AS MEASURED BY EQUILIBRIUM DIALYSIS

Transhydrogenase catalyses the transfer of reducing equivalents betwee n NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I prot ein) from Rhodospirillum rubrum and from Escherichia coli were overexp ressed and purified. Nucleotide binding to the domain I proteins was d etermined by equilibrium dialysis. NADH and its analogue, acetylpyridi ne adenine dinucleotide (reduced form), bound with relatively high aff inity (K-d = 32 mu M and 120 mu M, respectively, for the R. rubrum pro tein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitte rionic buffers, and at pH 7.5 in sodium phosphate buffer. NAD(+) bound with lower affinity (K-d = 300 mu M) NADPH bound only very weakly (K- d > 1 mM).Using a centrifugation procedure, Yamaguchi and Hatefi [Yama guchi, M. & Hatefi, Y. (1993) J. Biol. Chem. 268, 17871-17877] found t hat mitochondrial transhydrogenase, and a proteolytically derived doma in I fragment from that enzyme, bound one NADH per dimer. They suggest ed that this result implied half-of-the-sites reactivity for the inter action between the nucleotide ligand and the protein. However, our stu dies on both the E. coli and the R. rubrum recombinant transhydrogenas e domain I proteins using equilibrium dialysis show that the binding s toichiometry for both NADH and the reduced form of acetylpyridine aden ine dinucleotide (AcPdADH) is two nucleotides per dimer: no interactio n between the monomeric units is evident. Reasons for the discrepancie s between the work on bacterial and mitochondrial transhydrogenases ar e discussed.