Activity equilibria of the thiol-modulated chloroplast H+ -ATPase as a function of the proton gradient in the absence and presence of ADP and arsenate

The 9-aminoacridine fluorescence signal was calibrated for quantitative determination of transmembrane delta pH of isolated thylakoids by imposing defined phosphate potentials at static head in the absence of delta psi. This technique was subsequently employed in the investigation of actual equilibrium activities of the thiol-modulated H+ -ATPase as a function of delta pH in the absence and presence of ADP and arsenate which was used as a substitute for phosphate. Significant quantitative and qualitative differences were observed depending on the compounds present in the medium. In the presence of ADP alone, virtually no ATPase activity was detected at delta pH values less than 3; the sigmoidal dependence of activity on intrathylakoidal H+ concentration indicated positive cooperativity. In contrast, in the presence of arsenate (+/- ADP) substantial activity was found at much lower proton gradients and a different [Hin+] dependence was observed. In the absence of both, ADP and arsenate, an intermediate activity -delta pH relationship was obtained. The equilibrium levels of tightly bound ADP (+/- arsenate) as function of delta pH were essentially inverse to ATPase activity, indicating the significance of ADP and arsenate (= phosphate) binding for enzyme control. A model was established which describes the process of activation by maximally three consective reversible protonation reactions taking place from the lumen phase of the thylakoid, and the interaction of ADP and arsenate/phosphate from the medium phase at defined protonation stages of the enzyme. This model explains the experimental findings in quantitative manner and allows for relevant results reported in the literature. The model proposes a simple interpretation of active and inactive states of the chloroplast H+ -ATPase.