Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the CuII 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 CuII 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 CuII 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 CuII site in this form. Simulation of the 14N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the CuII 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 CuII site of PcoC.
Copper transport Copper proteins Electron paramagnetic resonance Electron spin echo envelope modulation Hyperfine sublevel correlation experiment
Electron paramagnetic resonance
Electron spin echo envelope modulation
Hyperfine sublevel correlation experiment
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We thank Australian Research Council for financial support via grant A29930204. S.C.D. and K.J.B. are funded in part by the National Health and Medical Research Council.