THE RATIO OF PROTONS TRANSLOCATED HYDRIDE ION EQUIVALENT TRANSFERRED BY NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE IN CHROMATOPHORES FROM RHODOSPIRILLUM-RUBRUM

The reduction of acetylpyridine adenine dinucleotide (AcPdAD+, an NAD analogue) by NADPH, in chromatophores treated with valinomycin, was a ccompanied by alkalinisation of the external medium, as measured by th e absorbance change of added cresol red, a simple, non-binding pH indi cator. Experiments with a stopped-flow spectrophotometer showed that i nitial (linear) rates of alkalinisation persisted for 1 - 2s. From the results of experiments in which H+ uptake was driven by a series of s hort flashes of light, the dependence of the outward proton leak on th e extent of H+ uptake was established. Thus, the proton leak was subtr acted from the initial rate of alkalinisation during transhydrogenatio n to give the true proton-uptake rate. The correction factor was usual ly about 10%. The ratio of protons translocated/H transferred from NAD PH to AcPdAD+ (the H+/H- ratio) was 0.60 +/- 0.06. The transhydrogenat ion 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 Hconsumption linked to the regeneration system was observed and permitt ed internal checks on the calibration of the cresol red absorbance cha nges. After correction for the proton leak and scalar proton uptake, a n H+/H- ratio of 0.60 +/- 0.30 was calculated from the initial rates. The water-soluble polypeptide of transhydrogenase (Th(s)) was washed f rom a sample of chromatophores to inhibit transhydrogenation activity and the accompanying H+ uptake. Re-addition of purified Th(s) to deple ted chromatophores led to recovery of transhydrogenation activity and of H+ uptake. In this reconstituted system the H+/H- was similar to th at in the native membranes. These results make it unlikely that the H/H- ratio is artefactually low because chromatophores have a populatio n of transhydrogenase which is not coupled to proton translocation. Fu rther evidence that the mechanistic H+/H- ratio of chromatophore trans hydrogenase 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 alka linisation was fitted by the sum of H+ uptake (the rate of transhydrog enation multiplied by the H+/H- ratio) plus the H+ leak, when the rati o 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. For mechanisms in which the translocated protons are directly involved in the chemical transformatiom, an H+/H- ratio of less than 1 is only pos sible if specificities of substrate binding or ligand conduction are n ot absolute or if barriers to protonation/deprotonation are not comple te, i.e. if the enzyme slips. If proton translocation is indirectly co upled to chemical transformation by way of conformational changes, the n an H+/H- ratio of 0.5 can be explained if transhydrogenase operates as a dimer with interacting catalytic sites