Stoichiometry of reduction and phosphorylation during illumination of intact chloroplasts

Abstract

1. 1. Intact chloroplasts which had retained their envelopes and were capable of photoreducing CO 2 at rates ranging from 70 to 240 μmoles·mg −1 chlorophyll·h −1 showed a large increase in their endogenous levels of ATP and NADPH on illumination in the absence of electron acceptors. At low light intensities, addition of 3-phosphoglycerate, whose reduction consumes ATP and NADPH in an equimolar ratio, drastically decreased the levels both of chloroplast ATP and NADPH. Only at light intensities not far from light saturation for 3-phosphoglycerate reduction did ATP become available in excess of NADPH, as revealed by the different response of the endogenous levels of ATP and NADPH to addition of 3-phosphoglycerate. 2. 2. Addition of bicarbonate to preilluminated chloroplasts or reduction of the light intensity during photosynthetic CO 2 reduction decreased only the ATP, not the NADPH levels, indicating a rate limitation of CO 2 reduction by ATP. 3. 3. Quantum requirements for the reduction of 3-phosphoglycerate or oxaloacetate at low light intensities were consistently close to 4. For the reduction of CO 2 they were never significantly below 12 and usually much higher. As the reduction of CO 2 requires twice as much reduced pyridine nucleotide as that of 3-phosphoglycerate or oxaloacetate, this indicates that the ATP generated during pyridine nucleotide reduction is insufficient to drive CO 2 reduction. 4. 4. Dithiothreitol improved significantly quantum yields of CO 2 reduction, but not of 3-phosphoglycerate reduction. It also stimulated the reduction of oxygen (Mehler reaction) by intact or broken chloroplasts. 5. 5. The light-dependent increase of both chloroplast NADPH and ATP was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at concentrations which are ineffective in suppressing Photosystem I-dependent cyclic photophosphorylation of broken chloroplasts. 6. 6. From the data it is concluded that not much more than one molecule of ATP is formed in intact chloroplasts during the transport of two electrons from water to NADP +. Coupling of phosphorylation to electron transport appeared to be flexible, not tight. The extra ATP needed for the reduction of CO 2 was produced by an independent photochemical reaction presumably reducing oxygen.