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

We have recorded multi-frequency EPR spectra of ⁶³Cu- and ⁶⁵Cu-labeled, water-soluble Cu_A-protein from the cytochrome ba₃ 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.