Reaction Mechanism of Chloroplastic Ascorbate Peroxidase Based on the Crystal Structure

Abstract

Ascorbate peroxidase (APX) isoenzymes are known to play an important role in scavenging H₂O₂ in higher plants. APX utilizes ascorbate (AsA) as a specific electron-donor in the scavenging reaction. The object of this report is to elucidate the reaction mechanism of APX by the X-ray structure analysis and the single-crystal microspectroscopy. The crystals of APX were soaked in the reservoir solution containing 10 μM H₂O₂ or a mixture of H₂O₂ and 100 mM AsA and frozen rapidly at 100 K. Diffraction data were collected at SPring-8. The side-chain of Arg, one of the catalytic residues, was found to be shifted toward the ferryl-oxygen in the crystal soaked in H₂O₂. The electron density of the crystal soaked in H₂O₂ and AsA showed the location and orientation of AsA. The structural features and absorption spectra provided invaluable information for the reaction mechanism of APX.