Reaction pathways involved in the production of hydroxyl radicals in thylakoid membrane: EPR spin-trapping study

Abstract

It has been suggested that both free metals and reduced ferredoxin (Fd) participate in the light-induced production of hydroxyl radicals (OH˙) in thylakoid membranes of chloroplasts. The most direct evidence for the involvement of Fd in OH˙ formation under physiological conditions was reported by Jakob and Heber (Plant Cell Physiol., 1996, 37, 629–635), who used the oxidation of dimethylsulfoxide to methane sulfinic acid as an indicator of OH˙ production. We confirmed their conclusions using a more sensitive and reliable EPR spin-trapping method and extended their work by additional findings. Free metal-dependent and ferredoxin-dependent OH˙ production was studied simultaneously and strong metal chelator Desferal was used to distinguish between these reaction pathways. The participation of protein-bound iron within photosystem I was confirmed by partial suppression of OH˙ generation in broken chloroplasts by methyl viologen. The enhancement in the production of OH˙ in thylakoid membranes by externally added ferredoxin can be considered as a straightforward evidence of the involvement of ferredoxin in OH˙ formation.