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 2 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 + / K ratio of 0.60 2 0.30 was calculated from the initial rates.The water-soluble polypeptide of transhydrogenase (Th,) was washed from a sample of chroinatophores to inhibit transhydrogenation activity and the accompanying H + uptake. Re-addition of purified Th, to depleted chromatophores led to recovery of transhydrogenation activity and of H i 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 '/Hratio) plus the H' leak, when the ratio was 0.6 but not when it was 1.0. rubrum ; CTparticles, chromatophore membranes depleted of the soluble transhydrogenase component by repeated washing; dp, proton electrochemical potential gradient; ApH, transmembrane pH gradient; d y , transmembrane electrical potential.

Structure and mechanism of proton‐translocating transhydrogenase

“…In early experiments to determine the value of x in Eq. 1, rates of transhydrogenation, and of proton leak, were both underestimated (sometimes producing fortuitously accurate ratios, as discussed in [37]). Recent measurements, performed on a much faster time scale to minimise errors, indicate that, for both the bacterial and mitochondrial proteins, one proton is translocated across the membrane per hydride transferred [38].…”

Proton‐translocating transhydrogenase and NAD‐ and NADP‐linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria

“…First, the H÷/H -ratio for H÷-Thase (x in Equ. (1)) is probably only in the range 0.5-1.0 [2,15]; the enzyme is not a very good proton pump. This is reflected, for example, in the fact that it is difficult to drive ATP formation by transhydrogenation from NADPH to NAD + even in well-coupled submitochondrial particles [16].…”

“…ratio is 0.5. The real value of x is expected to lie between 0.5 and 1 .O, the limits set by experiment [2,15]. It is evident that, through the action of transhydrogenase, the NADP pool would be predominantly reduced over the range of Ap that is usually estimated in isolated mitochondria (lS&200 mV [ 171) and that, thermodynamically, for measured concentrations of IC and KG of 0.02-0.2 and 0.2-2 mM [9,11,40], respectively, the redox potential of the NADP'/NADPH would be low enough to drive NADP-ICDH from KG to IC (see above).…”