INTERACTION OF NUCLEOTIDES WITH THE NAD(H)-BINDING DOMAIN OF THE PROTON-TRANSLOCATING TRANSHYDROGENASE OF RHODOSPIRILLUM-RUBRUM

Transhydrogenase catalyzes the reduction of NADP(+) by NADH coupled to the translocation of protons across a membrane. The polypeptide compo sition of the enzyme in Rhodospirillum rubrum is unique in that the NA D(H)-binding domain (called Th-s) exists as a separate polypeptide. Th -s was expressed in Escherichia coli and purified. The binding of nucl eotide substrates and analogues to Th-s was examined by one dimensiona l proton nuclear magnetic resonance (NMR) spectroscopy and by measurin g the quenching of fluorescence of its lone Trp residue. NADH and redu ced acetylpyridine adenine dinucleotide bound tightly to Th-s, whereas NAD(+), oxidized acetylpyridine adenine dinucleotide, deamino-NADH, 5 '-AMP and adenosine bound less tightly. Reduced nicotinamide mononucle otide, NADPH and 2'-AMP bound only very weakly to Th-s. 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-s (Dig gle, 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 l oses mobility when Th-s binds nucleotide; the reaction involves two st eps. This was more clearly evident, even for tight-binding nucleotides , when experiments were carried out at higher temperatures (37 degrees C), where the resonances of the mobile loop were substantially narrow er. The binding of adenosine was sufficient to initiate loop closure; the presence of a reduced nicotinamide moiety in the dinucleotide appa rently serves to tighten the binding. Two-dimensional 1(H) NMR spectro scopy of the Th-s-5'-AMP complex revealed nuclear Overhauser effect in teractions between protons of amino acid residues in the mobile loop ( including those in a Tyr residue) and the nucleotide. This suggests th at, in the complex, the loop has closed down to within 0.5 nm of the n ucleotide.