Photoproduction and Detoxification of Hydroxyl radicals in Chloroplasts and Leaves and Relation to Photoinactivation of Photosystems I and II

The light-dependent production of hydroxyl radicals (HO*) by thylakoids, chloroplasts and leaves of Spinacia oleracea was investigated using dimethylsulfoxide as HO' trapping agent. Maximum rates of HO' production by thylakoids as indicated by the formation of methane sulfinic acid were observed under aerobic conditions in the absence of added electron acceptors. They were higher than 2 /anol (mgChlh)1. Saturation of HO* production occurred at the low photon flux density of 100 /rniol m~2 s1. Trapping of HO* by dimethylsulfoxide suppressed, but did not eliminate light-dependent inactivation of PSI and II suggesting that HO' formation contributed to the photosensitivity of isolated thylakoids. DCMU inhibited HO' formation. Importantly, methylviologen decreased HO* formation in the absence, but stimulated it in the presence of Fe 3+. In intact chloroplasts, HO' formation became appreciable only after KCN had been added to inhibit effective H2O2 scavenging by ascorbate peroxidase. It was stimulated by ferrisulfate, but not by ferricyanide which does not penetrate the chloroplast envelope. Infiltrated spinach leaves behaved similar in principle to intact chloroplasts in regard to HO* formation but HO' production was very slow if detectable at all by the formation of methylsulfini c acid indicating effective radical detoxification. HO' formation is interpreted to be the result of a Fenton-type reaction which produces HO' in chloroplasts from H2O2 and reduced ferredoxin, when O2 is electron acceptor in the Mehler reaction and radical detoxification reactions are inhibited.