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

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.