title: Metal-catalyzed oxidation reactions and mass spectrometry: the roles of ascorbate and different oxidizing agents in determining Cu-protein-binding sites.
abstract: Further study has been made of metal-catalyzed oxidation (MCO) reactions and mass spectrometry as a method to determine the binding site of copper in metalloproteins. The role of ascorbate and a variety of oxidizing agents, including O2, H2O2, and S2O8(2-), have been investigated using Cu/Zn superoxide dismutase (SOD) as a model system. Ascorbate is found to play two competing roles in the MCO reactions. It reduces Cu(II), which initiates and maintains the generation of reactive oxygen species, and it scavenges radicals, which helps to localize oxidation products to amino acids near the metal center. An ascorbate concentration of 100 mM is found to be optimal with regard to localizing oxidation products to only the Cu-binding residues (His44, His46, His61, and His118) of Cu/Zn SOD. This concentration of ascorbate is very similar to the optimum concentration found in our previous studies of different Cu-binding proteins. Another notable result from this study is the observation that S2O8(2-) is more effective as an oxidant than O2 or H2O2 in the MCO reactions. Because S2O8(2-) is more stable in solution than H2O2, using it as an oxidizing agent results in much less nonspecific oxidation to the protein. The overall results of this study suggest that general MCO reaction conditions may exist for determining the metal-binding site of a wide range of Cu-binding proteins.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Juma D
authors.initials: JD
authors.lastname: Bridgewater
authors.forename: Richard W
authors.initials: RW
authors.lastname: Vachet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin
chemical_list: D002352:Carrier Proteins; C023548:copper-binding protein; D003300:Copper; D013482:Superoxide Dismutase; D014357:Trypsin; D001205:Ascorbic Acid