Interaction of Nucleotides with the NAD(H)-binding Domain of the Proton-translocating Transhydrogenase of Rhodospirillum rubrum

• Published in: The Journal of biological chemistry 1996-04, Vol.271 (17), p.10103-10108
• Main Authors: Bizouarn, T, Diggle, C, Quirk, P G, Grimley, R L, Cotton, N P, Thomas, C M, Jackson, J B
• Format: Article
• Language: English
• Subjects: Biological Transport; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; NAD - analogs & derivatives; NAD - metabolism; NADP Transhydrogenases - metabolism; Oxidation-Reduction; Rhodospirillum rubrum; Rhodospirillum rubrum - enzymology; Spectrometry, Fluorescence; Tryptophan - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.17.10103

Transhydrogenase catalyzes the reduction of NADP by NADH coupled to the translocation of protons across a membrane. The polypeptide composition of the enzyme in Rhodospirillum rubrum is unique in that the NAD(H)-binding domain (called Th ) exists as a separate polypeptide. Th was expressed in Escherichia coli and purified. The binding of nucleotide substrates and analogues to Th was examined by one-dimensional proton nuclear magnetic resonance (NMR) spectroscopy and by measuring the quenching of fluorescence of its lone Trp residue. NADH and reduced acetylpyridine adenine dinucleotide bound tightly to Th , whereas NAD , oxidized acetylpyridine adenine dinucleotide, deamino-NADH, 5′-AMP and adenosine bound less tightly. Reduced nicotinamide mononucleotide, NADPH and 2′-AMP bound only very weakly to Th . The difference in the binding affinity between NADH and NAD indicates that there may be an energy requirement for the transfer of reducing equivalents into this site in the complete enzyme under physiological conditions. Earlier results had revealed a mobile loop at the surface of Th (Diggle, C., Cotton, N. P. J., Grimley, R. L., Quirk, P. G., Thomas, C. M., and Jackson, J. B. (1995) Eur. J. Biochem. 232, 315-326); the loop loses mobility when Th binds nucleotide; the reaction involves two steps. This was more clearly evident, even for tight-binding nucleotides, when experiments were carried out at higher temperatures (37°C), where the resonances of the mobile loop were substantially narrower. The binding of adenosine was sufficient to initiate loop closure; the presence of a reduced nicotinamide moiety in the dinucleotide apparently serves to tighten the binding. Two-dimensional 1 H NMR spectroscopy of the Th -5′-AMP complex revealed nuclear Overhauser effect interactions between protons of amino acid residues in the mobile loop (including those in a Tyr residue) and the nucleotide. This suggests that, in the complex, the loop has closed down to within 0.5 nm of the nucleotide.