Quantitative relationships between phosphorylation, electron flow, and internal hydrogen ion concentrations in spinach chloroplasts.

1. Further evidence that the uptake of [14C]hexylamine, determined by centrifugal filtration of spinach chloroplast thylakoids through silicone fluid layers, gives precise estimations of light-induced H+ concentration gradients (deltapH) is presented. DeltapH was independent of the amount of thylakoids used or of the concentration of hexylamine. Moreover, hexylamine uptake was sensitive to the osmolarity of the suspending medium. 2. Internal H+ concentration ([H+]in) is proportional to the rate of electron flow when light intensity was used to vary these parameters. Proportionality was still observed in the presence of 0.1 and 1.0 muM gramicidin D. When, however, [H+]in and electron flow were varied by increasing the concentration of gramicidin D, at constant light intensity the rate of electron flow was approximately proportional to 1/[H]in. 3. The phosphorylation efficiency (P/e2 ratio) falls with decreasing light intensity or increasing concentrations of the phosphorylation inhibitor, 4'-deoxyphlorizin. The proportionality between the rate of electron flow and [H+]in allows the calculation of the rate of nonphosphorylating (basal) electron flow if [H+]in under phosphorylating conditions is known. The contribution of basal electron flow, a consequence of passive efflux of H+ from the thylakoids, to the overall rate of electron flow increases as the rate of phosphorylation decreases. P/e2 ratios calculated using rates of electron flow from which the basal component has been subtracted are constant. A calculated P/e2 ratio of about 1.3 is obtained. 4. It is shown that the reciprocal of the phosphorylation efficiency should be proportional to 1/[H+]in2 when these parameters are varied using light intensity. This relationship was verified and provided an estimate of the P/e2 at infinite [H+]in. This value was 1.3. These results provide further evidence that a H+ electrochemical gradient serves to couple photophosphorylation to electron flow and that the rate of phosphorylation is proportional to [H+]in3. That is, three H+ are translocated out of thylakoids for each adenosine triphosphate formed.