STOICHIOMETRIES AND ENERGETICS OF PROTON TRANSLOCATION COUPLED TO ELECTRON-TRANSPORT IN CHLOROPLASTS

We have investigated the stoichiometry of electron transport-driven proton uptake from the external medium and proton release into thylakoid vesicles of chloroplasts during single-turnover flash illumination. Proton uptake from the external medium was measured using a glass pH-electrode. For each electron originating from the oxidation of water by Photosystem II and ultimately forming H2O2 via the photoreduction of methyl viologen, approx. 2.7 proteons were taken up from the external medium. For each electron originating from the oxidation of duroquinol by the cytochrme b/f complex and ultimately forming H2O2 via methyl viologen under conditions in which water oxidation was inhibited by diuron, approx. 1.7 protons were taken up from the external medium. It is presumed that for each electron involved in the formation of H2O2 that one protein is taken up. Therefore, these results indicate that the electron-transfer reaction from H2O to Photosystem I involves the uptake of approx. 1.7 protons per electron, and that in the reaction from duroquinol to Photosystem I approx. 0.7 protons per electron are taken up. Proton release within the thylakoid vesicles was measured spectrophotometrically using the pH-sensitive dye neutral red. Flash-induced absorbance changes due to neutral red indicated that for each proton released within the thylakoid vesicle due to water oxidation approx. 1.9 protons appear inside the vesicle as a result of quinol oxidation. The flash-induced formation of ATP was determined by the incorporation of 32Pi into ATP. The initiation of flash-induced ATP formation by Photosystem II operating alone required nearly twice as many flashes as were necessary when the Photosystem I-dependent turnover of the cytochrome b/f complex was responsible for proton accumulation, and nearly three times more than when Photosystem II and the cytochrome b/f complex operated together. This is taken to indicate that both of the reactions that result in proton accumulation within the vesicle contribute energetically to ATP synthesis and contribute in proportion to their measured H+/e- ratio. All of these flash-induced measurements, proton uptake from the external medium, proton release inside the vesicle, and the filling of the energetic threshold pool required for the onset of ATP synthesis, demonstrate that for each electron transferred from water through the cytochrome b/f complex to Photosystem I nearly three protons are accumulated within the thylakoid membrane. Thus, these data indicate that oxidiation of quinol by the cytochrome b/f complex is able to support the translocation of two protons per electron during flash energization.