1. H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H(+)-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.
I . The activity of NAD(P)+ transhydrogenase in chromatophores of Rhodobacter capsulntus relaxed from a high rate during illumination to a lower rate after darkening with a half-time of approximately 100 ms.2. The dissipative ionic current flowing across the chromatophore membrane was increased in the presence of transhydrogenase substrates. This is attributed to proton current through the transhydrogenase enzyme. Subject to the assumption that transhydrogenase does not conduct in the absence of nucleotide substrates, the ratio of protons translocated across the membrane per hydride ion transferred was 0.4 0.5. Within the error and uncertainties in the calibration procedure, this ratio may be consistent with a stoichiometry of one but higher values seem unlikely. The ratio of hydride ion transferred in the transhydrogenase to electrons transferred through the cyclic electron transport system was approximately 0.2.3. The K%P values for the transhydrogenase substrates were determined for chromatophores in illuminated and darkened suspensions over a range of pH. These values are discussed in relation to the equivalent parameters reported for mitochondria transhydrogenase [Rydstrom, J. (1977) Biochim. Biophys. Actu 255, 9641 -96461 and were used to calculate the concentrations of substrates which effectively saturate the enzyme. 4. At substrate concentrations which were in excess of 8 x Kkpp the dependence of transhydrogenase rate on the value of the membrane potential (zero pH gradient) was determined at pH 6.3, 6.9, 7.6 and 9.0. The relation was similar at pH 6.9 and 7.6. At alkaline pH the apparent threshold in the relation became more prominent as it was shifted to slightly higher values of membrane potential. At acid pH a shift in the opposite direction diminished the apparent threshold and saturation at high membrane potential became more dominant. We use these data in an attempt to discriminate between two models of energy transduction: (a) the driving force exerted by the membrane potential is mediated by a pH gradient formed through the operation of a proton well in the transhydrogenase; (b) the membrane potential increases a rate constant for charge translocation through transhydrogenase by decreasing the effective height of the Eyring barrier for charge transfer across the membrane through the enzyme. The second model leads to a more simple description than the first of the pH dependence of transhydrogenase rate on membrane potential. 5. Divalent cations at very low concentrations ( M 0.1 mM) or monovalent cations at higher concentrations ( M 50 mM) stimulate considerably the transhydrogenase activity in chromatophores. The effect of monovalent and divalent cations on the relationship between the rate of transhydrogenase and membrane potential suggests that they do not influence the reaction by altering the membrane-potential-dependent rate constant.The nicotinamide adenine dinucleotide (pyridine nucleotide) transhydrogenase in the membranes of mitochondria and many bacteria is driven towards reduction o...
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