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

Abstract:

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.