Multi-frequency EPR Evidence for a Binuclear Cu A Center in Cytochrome c Oxidase: Studies with a 63Cu- and 65Cu-Enriched, Soluble Domain of the Cytochrome ba3 Subunit II from Thermus thermophilus

Abstract

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble Cu A-protein from the cytochrome ba 3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent g z ∼2.18 and g xy ∼2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that Cu A is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.