CO2 reduction by intact chloroplasts under a diminished proton gradient

9-Aminoacridine has been used to monitor the intrathylakoid pH of photosynthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO2 reduction. However, low concentrations of uncoupling amines such as NH3 or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO2-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) OR NIGERICIN, WHICH INHIBITED CO2 reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH4C1, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed.