Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F0F1 (CF0F1, EC 3.6.3.34) with an Fe2+/H2O2/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H2O2, by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS–PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF0F1 thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF0F1 inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF0F1.