Thermodynamics and kinetics of photophosphorylation in bacterial chromatophores and their relation with the transmembrane electrochemical potential difference of protons.

Abstract

The extent of the protonmotive force (transmembrane ΔH+/F) developed in bacterial chromatophores from Rhodopseudomonas capsulata under continous illumination has been determined with measurements of the quenching of 9-aminoacridine fluorescence and the red band shift of carotenoids. The extent of the membrane potential generated in the dark with K+ pulses in valinomycin-treated chromatophores has been evaluated theoretically and the validity of the calibration curves of the carotenoid signals, obtained by this procedure, has been analyzed consequently. The maximal affinity of the ATP synthesis reaction attainable by chromatophores at high light intensities has been measured and compared with the level of the protonmotive force; this analysis has been extended to conditions of partial uncoupling by a variety of agents with a different mode of action. The results show a parallel decrease of the two parameters and indicate that in most cases a stoichiometry of 2 H+ per ATP is sufficient to account for the force ratios obtained experimentally. However in extensively uncoupled membranes (Δp < 250 mV) photophosphorylation affinity reaches levels incompatible with a minimal stoichiometry of 2 H+ per ATP. The kinetics of light-induced ATP synthesis have been also correlated with the extent of the protonmotive force: a decrease of photophosphorylation linear with Δp is observed in all the uncoupled conditions tested with an apparent threshold of Δp between 120–180 mV, under which no net phosphorylation is detectable. When cyclic electron flow is limited, either by additions of antimycin A or by intensity of actinic light, a correlation between Δp and the rate of photophosphorylation markedly different from that observed in uncoupled conditions is obtained. The results, which are discussed in terms of the degree of coupling of energy transduction between proton gradients and ATP synthesis, using the approach of non-equilibrium thermodynamics, are not completely compatible with the postulates of the chemiosmotic coupling hypothesis in its minimal version.