Inactivation mechanism of ascorbate peroxidase at low concentrations of ascorbate; hydrogen peroxide decomposes Compound I of ascorbate peroxidase

Abstract

One of the characteristic properties of ascorbate peroxidase (APX), which distinguishes it from guaiacol peroxidase, Cyt c peroxidase and glutathione peroxidase, is the rapid inactivation of the enzyme under conditions where an electron donor is absent. When thylakoid-bound APX (tAPX) in 100 μM ascorbate was diluted 500-fold with an ascorbate-depleted medium, the enzymatic activity was lost with half time of about 15 s. The inactivation of tAPX was suppressed under anaerobic conditions and also by the addition of catalase, but it was unaffected by the addition of superoxide dismutase. These observations suggest that hydrogen peroxide at nanomolar levels, produced by autooxidation of ascorbate at lower than micromolar levels, might participate in the inactivation of tAPX. The participation of hydrogen peroxide was confirmed by the inactivation of tAPX upon incubation with hydrogen peroxide under anaerobic conditions. In the absence of ascorbate, the heme of the two-electron-oxidized intermediate of tAPX (designated Compound I) is decomposed by hydrogen peroxide. Thus, the instability of Compound I to hydrogen peroxide is responsible for the inactivation of APX when ascorbate is not available for Compound I and the enzyme cannot turnover.