title: Electron paramagnetic resonance characterization of the copper-resistance protein PcoC from Escherichia coli.
abstract: Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the Cu(II) site of the copper-resistance protein PcoC from Escherichia coli and certain variant forms. Electron spin echo envelope modulation (ESEEM) experiments confirm the presence of two histidine ligands, His1 and His92, at the Cu(II) site of wild-type PcoC, consistent with the available X-ray crystallographic data for the homolog CopC (67% sequence identity) from Pseudomonas syringae pv. tomato. The variants H1F and H92F each lack one of the histidine residues close to the Cu(II) site. The ESEEM data suggest that the surviving histidine residue remains as a ligand. The nA variant features an extra alanine residue at the N terminus, which demotes the His1 ligand to position 2. At least one of the two histidine residues is bound at the Cu(II) site in this form. Simulation of the (14)N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the Cu(II) sites of the wild-type, H92F and nA forms, while the H1F variant has two nitrogen ligands. The spectra of wild-type form can be fitted adequately with a 3N or a 4N model. The former is consistent with the crystal structure of the CopC homolog, where His1 acts as a bidentate ligand. The latter raises the possibility of an additional unidentified nitrogen ligand. The markedly different spectra of the H1F and nA forms compared with the wild-type and H92F proteins further highlight the integral role of the N-terminal histidine residue in the high-affinity Cu(II) site of PcoC.
authors.affiliation: Department of Pathology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia. sdrew@unimelb.edu.au
authors.forename: Simon C
authors.initials: SC
authors.lastname: Drew
authors.forename: Karrera Y
authors.initials: KY
authors.lastname: Djoko
authors.forename: Lianyi
authors.initials: L
authors.lastname: Zhang
authors.forename: Melissa
authors.initials: M
authors.lastname: Koay
authors.forename: John F
authors.initials: JF
authors.lastname: Boas
authors.forename: John R
authors.initials: JR
authors.lastname: Pilbrow
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Xiao
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Barnham
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Wedd
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D012680:Sensitivity and Specificity
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; C462541:PcoC protein, E coli