A change in ionization of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase regulates both hydride transfer and nucleotide release

Transhydrogenase couples the transfer of hydride-ion equivalents between NA D(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the mem brane. Coupling between transhydrogenation and proton translocation involve s changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single-turnover bu rst of hydride transfer between bound nucleotides; subsequent turnover is l imited by NADP(H) release. Stopped-flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site-directed mutagenesis. Two mutants with simila r catalytic properties to those of the wild-type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affect ed by nucleotide occupancy of dIII was different in the two mutants, and he nce two different procedures for determining the rate of nucleotide release were developed. The apparent first-order rate constants for NADP(+) releas e and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of t he protonated and unprotonated forms of dIII are consistent with those expe cted of intermediates in the NADP(H)-binding-change mechanism. The ionisabl e group might be a component of the proton-translocation pathway in the com plete enzyme.