Changes in the apparent Michaelis constant for ADP during photophosphorylation are consistent with delocalised chemiosmotic energy coupling

The apparent Michaelis constant (K_m) for ADP has been measured under various conditions of steady-state photophosphorylation in isolated thylakoid membranes. In addition, the steady-state ΔpH has been simultaneously estimated from the fluorescence quenching of 9-aminoacridine. The following results were obtained. (1) The standard procedure for estimating K_m, by increasing the concentration of ADP, progressively lowered the steady-state ΔpH, thereby introducing an uncontrolled system variable into the K_m analysis. This has the effect of lowering the apparent K_m measured. (2) Lowering the light intensity lowered the observed K_m, and addition of uncouplers increased the observed K_m. The ability of uncouplers to increase K_m was enhanced at lowered light intensities. In contrast, the effect of lowered light intensity on the observed K_m was diminished and then reversed under progressively more uncoupled conditions. (3) The addition of energy-transfer inhibitors caused an increase in the observed K_m for ADP. (4) All of the observations are qualitatively predicted by a mathematical model based on simple delocalised chemiosmotic energy coupling and Michaelis-Menten kinetics for the chloroplast ATPase with respect to ADP. It is concluded that the complex behaviour of the apparent K_m for ADP under different conditions arises because ΔpH is an uncontrolled variable during the K_m analysis and that the results are entirely consistent with a model of delocalised chemiosmotic energy coupling.