Iron-mediated inhibition of H+-ATPase in plasma membrane vesicles isolated from wheat roots

The mechanisms of iron-mediated inhibition of the H+-ATPase activity of plasma membrane (PM) vesicles isolated from wheat roots were investigated. Both FeSO4 and FeCl3 significantly inhibited PM H+-ATPase activity, and the inhibition could be reversed by the addition of the metal ion chelator EDTA-Na2 or a specific Fe2+ chelator, indicating that the inhibitory effect was due to specific action of Fe2+ or Fe3+. Measurement of the extent of lipid peroxidation showed that oxidative damage on the PM caused by Fe2+ or Fe3+ seemed to be correlated with the inhibition of PM H+-ATPase activity. However, prevention of lipid peroxidation with butylated hydroxytoluene did not affect iron-mediated inhibition in the PM H+-ATPase, suggesting that the inhibition of the PM H+-ATPase was not a consequence of lipid peroxidation caused by iron. Investigation of the effects of various reactive oxygen species scavengers on the iron-mediated inhibition of H+-ATPase activity indicated that hydroxyl radicals (*OH) and hydrogen peroxide (H2O2) might be involved in the Fe2+-mediated decrease in PM H+-ATPase activity. Moreover, iron caused a decrease in plasma protein thiol (P-SH), and Fe3+ brought a higher degree of oxidation in thiol groups than Fe2+ at the same concentration. Modification of the thiol redox state in the PM suggested that reducing thiol groups were essential to maintain PM H+-ATPase activity. Incubation of the specific thiol modification reagent 5,5-dithio-bis(2-nitrobenzoic acid) with the rightside-out and inside-out PM revealed that thiol oxidation occurred at the apoplast side of the PM. Western blotting analysis revealed a decrease in H+-ATPase content caused by iron. Taken together, these results suggested that thiol oxidation might account for the inhibition of PM H+-ATPase caused by iron, and that *OH and H2O2 were also involved in Fe2+-mediated inhibition.