PROPERTIES OF THE PURIFIED, RECOMBINANT, NADP(H)-BINDING DOMAIN-III OF THE PROTON-TRANSLOCATING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM

Transhydrogenase comprises three domains. Domains I and III are periph eral to the membrane and possess the NAD(H)- and NADP(H)-binding sites , respectively, and domain II spans the membrane, Domain III of transh ydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli. and purified. The purified protein was associated wi th substoichiometric quantities of tightly bound NADP(+) and NADPH. Fl uorescence spectra of the domain III protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nu cleotide are spatially close. The rate constants for NADP(+) release a nd NADPH release from domain III were 0.03 s(-1) and 5.6X10(-4)s(-1), respectively In the absence of domain II a mixture of the recombinant domain III protein, plus the previously described recombinant domain I protein, catalysed reduction of acetylpyridine - adenine dinucleotide (AcPdAD(+)) by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP(+) from domain III, Similarly, the mixt ure catalysed reduction of thio-NADP(+) by NADH (forward transhydrogen ation) at a rate limited by release of thio-NADPH from domain III. The mixture also catalysed very rapid reduction of AcPdAD(+) by NADH, pro bably by way of a cyclic reaction mediated by the tightly bound NADP(H ). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain III and vice versa indicated (a) t hat during reduction of AcPdAD(+) by NADPH, a single domain I protein can visit and transfer H- equivalents to about 60 domain III proteins during the time taken for a single domain III to release its NADP(+), whereas (b) the cyclic reaction is rapid on the timescale of formation and breakdown of the domain I . III complex. The I ate of the hydride transfer reaction was similar in the domain I . III complex to that i n the complete membrane-bound transhydrogenase, but the fates of forwa rd and reverse transhydrogenation were much slower in the I . III comp lex due to the greatly decreased rates of release of NADP(+) and NADPH . It is concluded that, in the complete enzyme, conformational changes in the membrane-spanning domain II, which result from proton transloc ation, lead to changes in the binding affinity of domain III for NADP( -) and for NADPH.