INTACT CHLOROPLASTS DISPLAY PH-5 OPTIMUM OF O2-REDUCTION IN THE ABSENCE OF METHYL VIOLOGEN - INDIRECT EVIDENCE FOR A REGULATORY ROLE OF SUPEROXIDE PROTONATION

The pH-dependence of light-driven O2-reduction in intact spinach chloroplasts is studied by means of chlorophyll fluorescence quenching analysis and polarographic O2-uptake measurements. Most experiments are carried out in presence of KCN, which blocks activities of Calvin cycle, ascorbate peroxidase and superoxide dismutase. pH is varied by equilibration with external buffers in presence of nigericin. Vastly different pH-optima for O2-dependent electron flow are observed in the presence and absence of the redox catalyst methyl viologen. Both fluorescence quenching analysis and O2-uptake reveal a distinct pH 5 optimum of O2-reduction in the absence of methyl viologen. In the presence of this catalyst, O2-reduction is favoured in the alkaline region, with an optimum around pH 8, similar to other types of Hill reaction. It is suggested that in the absence of methyl viologen the extent of irreversibility of O2-reduction is determined by the rate of superoxide protonation. This implies that O2-reduction takes place within the aprotic phase of the thylakoid membrane and that superoxide-reoxidation via oxidized PSI donors competes with protonation. Superoxide protonation is proposed to occur at the internal surface of the thylakoid membrane. There is no competition between superoxide reoxidation and protonation when in the presence of methyl viologen the site of O2-reduction is shifted into the protic stroma phase. In confirmation of this interpretation, fluorescence measurements in the absence of KCN reveal, that non-catalysed O2-dependent electron flow is unique in being stimulated by the transthylakoidal pH-gradient. On the basis of these findings a major regulatory role of O2-dependent electron flow under excess light conditions is postulated.