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 chlor oplasts is studied by means of chlorophyll fluorescence quenching anal ysis and polarographic O2-uptake measurements. Most experiments are ca rried out in presence of KCN, which blocks activities of Calvin cycle, ascorbate peroxidase and superoxide dismutase. pH is varied by equili bration with external buffers in presence of nigericin. Vastly differe nt pH-optima for O2-dependent electron flow are observed in the presen ce and absence of the redox catalyst methyl viologen. Both fluorescenc e quenching analysis and O2-uptake reveal a distinct pH 5 optimum of O 2-reduction in the absence of methyl viologen. In the presence of this catalyst, O2-reduction is favoured in the alkaline region, with an op timum around pH 8, similar to other types of Hill reaction. It is sugg ested that in the absence of methyl viologen the extent of irreversibi lity of O2-reduction is determined by the rate of superoxide protonati on. This implies that O2-reduction takes place within the aprotic phas e of the thylakoid membrane and that superoxide-reoxidation via oxidiz ed PSI donors competes with protonation. Superoxide protonation is pro posed to occur at the internal surface of the thylakoid membrane. Ther e is no competition between superoxide reoxidation and protonation whe n in the presence of methyl viologen the site of O2-reduction is shift ed into the protic stroma phase. In confirmation of this interpretatio n, fluorescence measurements in the absence of KCN reveal, that non-ca talysed O2-dependent electron flow is unique in being stimulated by th e transthylakoidal pH-gradient. On the basis of these findings a major regulatory role of O2-dependent electron flow under excess light cond itions is postulated.