title: Distinct oxidative cleavage and modification of bovine [Cu- Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule.
abstract: We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu-Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction.
authors.affiliation: Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, White Oak, MD 20993, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uehara
authors.affiliation: Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, White Oak, MD 20993, USA.
authors.forename: Shen
authors.initials: S
authors.lastname: Luo
authors.affiliation: Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, White Oak, MD 20993, USA.
authors.forename: Baikuntha
authors.initials: B
authors.lastname: Aryal
authors.affiliation: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA.
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
authors.affiliation: Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, White Oak, MD 20993, USA. Electronic address: ashutosh.rao@fda.hhs.gov.
authors.forename: V Ashutosh
authors.initials: VA
authors.lastname: Rao
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003300:Copper / Q000737:chemistry* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D050050:Protein Carbonylation; D011487:Protein Conformation; D000072105:Superoxide Dismutase-1 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003300:Copper; D006861:Hydrogen Peroxide; D000072105:Superoxide Dismutase-1; D001205:Ascorbic Acid
keywords: Ascorbic acid; Carbonylation; Copper; Mass spectrometry; Metal catalyzed oxidation; SOD