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 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 Escherichia coli were overexpressed and purified. Nucleotide 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 (Kd = 32 microM and 120 microM, 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+ bound with lower affinity (Kd = 300 microM). NADPH bound only very weakly (Kd > 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M. & Hatefi, Y. (1993) J. Biol. Chem. 268. 17871-17877] found that mitochondrial transhydrogenase, and a proteolytically derived domain I fragment from that enzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-site 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 proteins using equilibrium 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 monomeric units is evident. Reasons for the discrepancies between the work on bacterial and mitochondrial transhydrogenases are discussed.
Bizouarn
T
T
School of Biochemistry, University of Birmingham, England.
Diggle
C
C
Jackson
J B
JB
D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics
D003067:Coenzymes; D009711:Nucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009243:NAD; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases