Using metal-catalyzed oxidation reactions and mass spectrometry to identify amino acid residues within 10 A of the metal in Cu-binding proteins.
Metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) can be used together to determine the amino acids bound to Cu in a metalloprotein. Selective oxidation of only amino acids bound to Cu during the MCO/MS approach relies on proper choice of the types and concentrations of the reducing and oxidizing agents. We show here that if these MCO reagent concentrations are "detuned" or varied slightly from optimal conditions, nonmetal-bound amino acids close to Cu can also be oxidized in a controlled manner. Using Cu/Zn superoxide dismutase as a model system, we demonstrate that amino acids up to 10 A from Cu can be modified as long as they are readily accessible to diffusing reactive oxygen species. UV/VIS spectroscopy and measurements of oxidation kinetics provide evidence that the protein's structural integrity around Cu is maintained during the detuned MCO reactions. Because this method can identify amino acids around Cu that have low solvent accessibility, it should complement other radical-based protein surface-mapping techniques.
Bridgewater
Juma D
JD
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Lim
Jihyeon
J
Vachet
Richard W
RW
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001400:Azurin / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003300:Copper / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000737:chemistry
D000596:Amino Acids; D008667:Metalloproteins; D001400:Azurin; D003300:Copper; D013482:Superoxide Dismutase