Studies on the Energy-coupling Sites of Photophosphorylation: II. Treatment of Chloroplasts with NH(2)OH Plus Ethylenediaminetetraacetate to Inhibit Water Oxidation while Maintaining Energy-coupling Efficiencies

Abstract

Artificial electron donors to photosystem II provide an important means for characterizing the newly discovered site of energy coupling near photosystem II. However, water oxidation must be completely abolished, without harming the phosphorylation mechanism, for these donor reactions and the associated phosphorylation to withstand rigorous quantitative analysis. In this paper we have demonstrated that treatment of chloroplasts with hydroxylamine plus EDTA at pH 7.5 in the presence of Mg(2+) followed by washing to remove the amine is a highly reliable technique for this purpose. The decline of the Hill reaction and the coupled phosphorylation during the treatment were carefully followed. No change in the efficiency of phosphorylation (P/e(2) 1.0-1.1) was observed until the reactions became immeasurable. Photosystem I-dependent reactions, such as the transfer of electrons from diaminodurene or reduced 2,6-dichlorophenolindophenol to methylviologen, and the associated phosphorylation were totally unaffected. It is clear that the hydroxylamine treatment is highly specific, with no adverse effect on the mechanism of phosphorylation itself. Benzidine photooxidation via both photosystems II and I in hydroxylamine-treated chloroplasts (electron acceptor, methylviologen; assayed as O(2) uptake) supports phosphorylation with the same efficiency as that observed for the normal Hill reaction (P/e(2) = 1.1). An apparent P/e(2) ratio of 0.6 was computed for the photooxidation of ascorbate.