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Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy

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Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu2+ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu2+ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.

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  1. The Euler angles as used in this work specify three consecutive rotations in the following order: (1) by φ around axis 3 (or z); (2) by θ around newly obtained axis 2 (or y); (3) by ψ around newly obtained axis 3 (or z).


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We thank Joshua Kittleson for assistance with sample preparation. The authors acknowledge the NSF funding (DBI-9604939) for construction and modification of the pulsed EPR spectrometer used in this work.

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Correspondence to Megan M. McEvoy.

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Astashkin, A.V., Raitsimring, A.M., Walker, F.A. et al. Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy. J Biol Inorg Chem 10, 221–230 (2005).

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