Characterisation of membrane vesicles from Paracoccus denitrificans and measurements of the effect of partial uncoupling on their thermodynamics of oxidative phosphorylation.

Abstract

1. Vesicles from Paracoccus denitrificans were prepared by applying an osmotic shock to spheroplasts derived from cells that had been grown anaerobically with succinate as carbon source and nitrate as electron acceptor. In the presence of either phenazinemethosulphate or N,N,N' N',-tetramethyl-p-phenylenediamine, the oxidation of isoascorbate supported the uptake of both S14CN- and 86Rb+ (in the presence of valinomycin), whereas NADH and succinate oxidation resulted only in S14CN- uptake. These observations show that the preparations contain both right-side-out and inside-out vesicles, and are related to the earlier proposal that the stimulation of an NADH-2,6-dichloroindophenol reductase activity by bee venom is an indicator of the proportion of right-side-out vesicles present. The implications impinge on previous conclusions [Burnell, J. N., John. P. and Whatley, F. R. (1975) Biochem. J. 150, 527-536 and FEBS Lett. 58, 215-218] about the mechanisms of sulphate and phosphate transport in P. denitrificans. 2. The relationship between the protonmotive force (delta p; transmembrane proton electrochemical gradient expressed in mV) and the phosphorylation potential (delta Gp) generated by vesicles from P. denitrificans has been studied as a function of the concentration of an uncoupler of oxidative phosphorylation. With either NADH or succinate as substrate, the uncoupler had a more pronounced effect on delta p than on delta Gp, so that the ratio delta Gp/F x delta p increased within a limited range of values of delta p close to the maximum. delta Gp/F x delta p was, however, approximately constant over the remaining range of delta p that was titrated. A fraction of 'highly coupled' vesicles, separated from the initial preparation by centrifugation through a Ficoll pad, showed similar titration behaviour. This demonstrated that heterogeneity within a vesicle preparation was not responsible for significant distortion of the true relationship between delta p and delta Gp. Values of delta p and delta Gp/F x delta p (H+/ATP) from 143-108 mV and 3.9-4.4, respectively, were determined when NADH was substrate, whereas with succinate, delta p ranged from 123-88 mV and delta Gp/F x delta p (H+/ATP) from 4.5-5.6. The variation in the value of delta Gp/F x delta p, which can be equated with a minimum value for the H+/ATP of the ATP synthase enzyme, is similar to, but less pronounced than, some of the data previously reported for mitochondria. Thus the observations with these bacterial vesicles, which represent an experimentally simpler system than mitochondria, might be taken as further evidence that measurements of the bulk phase delta p might not truly reflect the driving force for ATP synthesis sensed by the ATP synthase enzyme. However, other explanations that would make the data consistent with a chemiosmotic mechanism cannot be eliminated...