Uncoupling and energy transfer inhibition of photophosphorylation by sulfhydryl reagents.

The action of the sulfhydryl reagents, o-iodosobenzoate and 2,2’-dithiobis(5-nitropyridine), on photophosphorylation by spinach chloroplast thylakoids has been reevaluated. Both of these compounds were previously reported to be energy transfer inhibitors of photophosphorylation, provided the thylakoids were illuminated in their presence prior to assay. We show here that the treatment of thylakoids in the light with iodosobenzoate uncouples phosphorylation from electron flow. This treatment enhances nonphosphorylating electron transport and markedly decreases the efficiency of photophosphorylation. The light-induced transmembrane pH gradient is also diminished by exposure of thylakoids to iodosobenzoate in the light. Dithiobisnitropyridine has been found to act either as a light-dependent uncoupler or energy transfer inhibitor. At low concentrations of this reagent, illumination elicits uncoupling, whereas at higher concentrations, energy transfer inhibition is induced. The uncoupling by iodosobenzoate and by low concentrations of dithiobisnitropyridine is largely prevented by the prior incubation of thylakoids with N-ethylmaleimide in the dark. Under these conditions, N-ethylmaleimide was previously shown to react with a group on the y subunit and with a group on the E subunit of coupling factor 1. The effects of these sulfhydryl reagents on photophosphorylation are compared to those of maleimides, and a model for the inhibition of phosphorylation by these reagents is proposed. Cross-linking two sufhydryl groups within the y subunit of coupling factor 1, either directly by disulfide bond formation or by bifunctional maleimides, causes thylakoids to become proton-leaky and phosphorylation is uncoupled from electron flow. In contrast, modification of a sulfhydryl, which becomes exposed only in the light, by monofunctional reagents elicits energy transfer inhibition.