Structural changes in the active site of the chloroplastic ascorbate peroxidase during its H₂O₂-induced inactivation.

Abstract

APX isoforms localized in chloroplasts of higher plants are rapidly inactivated by attack of hydrogen peroxide if the second substrate ascorbate is depleted. This is the reason why chloroplasts are damaged under oxidative stresses. We studied the molecular mechanism of the H₂O₂-induced inactivation of chloroplastic APXs. We found chloroplastic APXs have amino acid sequences inserted in C-terminal half domain, which is not found in those of H₂O₂-tolerant APX isoforms. When this region was inserted into the corresponding site of a Galdieria APX, an H₂O₂-tolerant APX, it showed rapid inactivation by addition of H₂O₂ under depletion of ascorbate. This indicate that interaction between the catalytic site and this region is responsible for the inactivation of APX by H₂O₂. Furthermore, we found that the inactivation of the chloroplastic APX associated with covalent binding of the heme located at the catalytic site to the apoprotein.