ON PROPERTIES OF ASCORBATE PHOTOOXIDATION IN ISOLATED CHLOROPLASTS - EVIDENCE FOR 2 ATP SITES IN NONCYCLIC PHOTOPHOSPHORYLATION

Ascorbate photooxidation was studied in isolated spinach chloroplasts. The stoichiometry of the reaction is 1 μmole ascorbate oxidized per 1 μmole O2 taken up. The reaction is stimulated by the addition of an electron acceptor for light reaction I, like anthraquinone or methylviologen; it is inhibited by DCMU and other inhibitors of light reaction II and does not take place in light above 700 mμ. The rate of ascorbate photooxidation is controlled by the phosphorylating system, since it is stimulated by the addition of either the phosphorylating system or an uncoupler. Coupled ascorbate photooxidation is inhibited by DCCD, an inhibitor of ATP formation. Heated spinach chloroplasts, not able to evolve O2, still oxidize ascorbate coupled to ATP formation in a 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-sensitive reaction. It is concluded that ascorbate replaces water as electron donor for light reaction II and that both light reactions and the electron transport system between the two light reactions including one ATP site are participating in ascorbate photooxidation. The measured stoichiometry of ATP formation to ascorbate oxidation and O2 uptake is 0.5:1:1. By subtracting the basal rate of uncoupled electron flow in ascorbate photooxidation from the rate of coupled electron flow, the corrected stoichiometry of 1 mole ATP per 2 electrons transferred is obtained. By the same calculation 2 moles ATP are formed per 2 electrons transferred, if water is the electron donor in a Hill reaction. Therefore the results indicate that there may be an ATP site between water and pigment system II; this ATP site is not operating in ascorbate photooxidation. © 1969.