A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

Abstract

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO •) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H2O2-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H2O 2-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO• and DMPO in an H2O2-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H2O2. TEMPO• bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H2O2, both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H2O 2-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group. © 2007 The Authors.