The ratio of protons translocated/hydride ion equivalent transferred by nicotinamide nucleotide transhydrogenase in chromatophores from Rhodospirillum rubrum

Abstract

The reduction of acetylpyridine adenine dinucleotide (AcPdAD+, an NAD+ analogue) by NADPH, in chromatophores treated with valinomycin, was accompanied by alkalinisation of the external medium, as measured by the absorbance change of added cresol red, a simple, non-binding pH indicator. Experiments with a stopped-flow spectrophotometer showed that initial (linear) rates of alkalinisation persisted for 1-2s. From the results of experiments in which H+ uptake was driven by a series of short flashes of light, the dependence of the outward proton leak on the extent of H+ uptake was established. Thus, the proton leak was subtracted from the initial rate of alkalinisation during transhydrogenation to give the true proton-uptake rate. The correction factor was usually about 10%. The ratio of protons translocated/H- transferred from NADPH to AcPdAD+ (the H+/H- ratio) was 0.60 +/- 0.06. The transhydrogenation reaction between NAD+ and NADPH was measured in the presence of a regeneration system for NAD+ (pyruvate and lactate dehydrogenase). In addition to the accompanying proton-translocation reaction, scalar H+ consumption linked to the regeneration system was observed and permitted internal checks on the calibration of the cresol red absorbance changes. After correction for the proton leak and scalar proton uptake, an H+/H- ratio of 0.60 +/- 0.30 was calculated from the initial rates. The water-soluble polypeptide of transhydrogenase (Ths) was washed from a sample of chromatophores to inhibit transhydrogenation activity and the accompanying H+ uptake. Re-addition of purified Ths to depleted chromatophores led to recovery of transhydrogenation activity and of H+ uptake. In this reconstituted system the H+/H- was similar to that in the native membranes. These results make it unlikely that the H+/H- ratio is artefactually low because chromatophores have a population of transhydrogenase which is not coupled to proton translocation. Further evidence that the mechanistic H+/H- ratio of chromatophore transhydrogenase is less than 1 was provided by an analysis of the kinetics of alkalinisation of the medium during reduction of AcPdAD+ by NADPH. It was shown that the progress of the transhydrogenation-induced alkalinisation was fitted by the sum of H+ uptake (the rate of transhydrogenation multiplied by the H+/H- ratio) plus the H+ leak, when the ratio was 0.6 but not when it was 1.0. The results are discussed in terms of the possible mechanism of energy coupling by transhydrogenase.(ABSTRACT TRUNCATED AT 400 WORDS)