Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts

Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine $\text{ATP}\text{2}\text{e}$ ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a $\text{1}\text{1}$ ratio, was about 4 both at pH 7.6 and at 7.0. $\text{ATP}\text{2}\text{e}$ ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2.Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO₂. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities $\text{ATP}\text{2}\text{e}$ ratios were calculated to be between 1.1 and 1.4.Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg^?1 chlorophyll · h^?1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO₂. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated $\text{ATP}\text{2}\text{e}$ ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials $\text{ATP}\text{]}\text{ADP}\text{]}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$. Flexible coupling implies, that $\text{ATP}\text{2}\text{e}$ ratios should decrease with increasing phosphorylation potentials inside the chloroplasts.