Energetic factors affecting carbon dioxide fixation in isolated chloroplasts

Light- and HCO(3) (-)-saturated (10 millimolar) rates of O(2) evolution (120 to 220 micromoles O(2) per milligram chlorophyll per hour), obtained with intact spinach chloroplasts, are decreased up to 3-fold by changes in assay conditions such as omission of catalase from the medium, the use of high (>/=1 millimolar) inorganic phosphate, inclusion of NO(2) (-) as an electron acceptor, or bright illumination at low partial pressures of O(2). These inhibitions may be reversed by addition of uncoupling levels of NH(4)Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b(6) and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in DeltapH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO(3) (-)-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions.The rate of O(2) evolution is also decreased 3-fold when ambient CO(2) (0.63 millimolar HCO(3) (-) at pH 8.1) is used in place of saturating HCO(3) (-) and chloroplasts are illuminated aerobically with catalase and a low level (0.25 millimolar) of K(2)HPO(4). Only inhibitory effects are observed with additions of antimycin or NH(4)Cl. Under these conditions, excessive photophosphorylation or a large pH gradient does not limit the rate of photosynthesis.