TY - JOUR
T1 - Using Metal-Catalyzed Oxidation Reactions and Mass Spectrometry to Identify Amino Acid Residues Within 10 Å of the Metal in Cu-Binding Proteins
AU - Bridgewater, Juma D.
AU - Lim, Jihyeon
AU - Vachet, Richard W.
N1 - Funding Information:
This work was supported by a grant from the National Institutes of Health (RO1 GM075092).
PY - 2006/11
Y1 - 2006/11
N2 - 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 Å 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.
AB - 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 Å 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.
UR - http://www.scopus.com/inward/record.url?scp=33750299045&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33750299045&partnerID=8YFLogxK
U2 - 10.1016/j.jasms.2006.06.003
DO - 10.1016/j.jasms.2006.06.003
M3 - Article
C2 - 16872838
AN - SCOPUS:33750299045
SN - 1044-0305
VL - 17
SP - 1552
EP - 1559
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 11
ER -