Chloroplast membrane conformational changes measured by chemical modification

The chemical modification reagents iodoacetic acid (primarily sulfhydryl group directed) and acetic anhydride (primarily amino group directed) were used to monitor chloroplast thylakoid membrane conformational changes. The incorporation of [³H]-iodoacetate and [³H]acetic anhydride showed the following pattern: (i) There was an increased level of binding of iodoacetate in the light compared to the dark or light plus 2,4-dichlorophenyl-1,1-dimethyl urea (DCMU) conditions. A 30 to 50% increase, from about 1.0 to 1.3–1.5 nmol/mg of Chl in iodoacetate incorporation, was found; 30–50% less acetic anhydride was bound in the light than in the dark or light plus DCMU state, typical values being near 15 nmol of acetic anhydride bound/mg of Chl in the dark and 10 nmol/mg of Chl in the light, (ii) The incorporation pattern for both reagents indicated that Photosystem II-dependent proton release is required to elicit the differential binding. Evidence for this is: (a) Cyclic electron flow and proton accumulation, mediated by phenazine methosulfate in the presence of a Photosystem II inhibitor (DCMU), did not induce either the extra binding of iodoacetate or the decrease in binding of acetic anhydride; (b) in chloroplasts made deficient in water oxidation by NH₂OH treatment, electron flow from I^?, an alternate Photosystem II electron donor, to methyl viologen did not induce the differential binding, whereas with the proton-donating donor, diphenyl carbazide, Photosystem II electron flow did elicit the differential binding, (iii) Uncouplers of phosphorylation (nigericin plus valinomycin) had no affect on the differential binding of either reagent, consistent with the hypothesis that it is not simply a transmembrane proton gradient that potentiates the conformational change, but rather an intramembrane reaction between protons released by Photosystem II and certain membrane components. The lack of uncoupler effect also suggests that the conformational change does not involve the coupling factor complex, at least not in the same sense as for the coupling factor conformational changes detected by tritium exchange (I. J. Ryrie and A. T. Jagendorf, 1971, J. Biol. Chem.246, 582–588) or N-ethyl maleimide binding (R. E. McCarty et al., 1972, J. Biol. Chem.247, 3048–3051). (iv) The decrease in acetic anhydride binding in the light was independent of the structural state of the chloroplast. Stacked and unstacked (by low salt) grana membranes showed similar light-dependent decreases in acetic anhydride binding. The results with these modification reagents support earlier conclusions about a Photosystem II-linked conformational change based on work with diazonium benzenesulfonic acid (R. Giaquinta et al., 1975, Biochemistry14, 4392–4396).