title: 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 H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(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 H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). 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 H(2)O(2), 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 H(2)O(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.
authors.affiliation: Graduate School of Science and Technology, Kyoto Institute of Technology, Japan. sakito@kit.ac.jp
authors.forename: Sakihito
authors.initials: S
authors.lastname: Kitajima
authors.forename: Miyo
authors.initials: M
authors.lastname: Kurioka
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yoshimoto
authors.forename: Mayumi
authors.initials: M
authors.lastname: Shindo
authors.forename: Kenji
authors.initials: K
authors.lastname: Kanaori
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Tajima
authors.forename: Kenji
authors.initials: K
authors.lastname: Oda
mesh_terms: D060387:Ascorbate Peroxidases; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011422:Propionates / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D000461:Rhodophyta / Q000201:enzymology; D053719:Tandem Mass Spectrometry; D014026:Nicotiana / Q000201:enzymology
chemical_list: D007527:Isoenzymes; D011422:Propionates; D011994:Recombinant Proteins; D006418:Heme; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D003545:Cysteine