Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (O-1(2)center dot) in the intact and the Rieske-depleted cytochrome b(6)f complexes (Cyt b(6)f) from Bryopsis corticulans, as well as in the isolated Rieske Fe-S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b(6)f can be bleached by light-induced O-1(2)center dot, and that the 10; production can be promoted by D2O or scavenged by extraneous antioxidants such as L-histidine, ascorbate, beta-carotene and glutathione. Under similar experimental conditions, O-1(2)center dot was also detected in the Rieske-depleted Cyt b(6)f complex, but not in the isolated Rieske Fe-S protein. The results prove that Chl a cofactor, rather than Rieske Fe-S protein, is the specific site of O-1(2)center dot formation, a conclusion which draws further support from the generation of O-1(2)center dot with selective excitation of Chl a using monocolor red light. (C) 2009 Elsevier B.V. All rights reserved.