The EPR spectrum for Cu_B in cytochrome c oxidase

Incubation of cytochrome c oxidase (CcO) in its resting state in saturated ammonium sulfate, at room temperature overnight, gave EPR signals characteristic of a single Cu(II) center. From the g_|| and A_|| values it is concluded that this is a square-planar type 2 copper center, and superhyperfine splitting shows the presence of three nearly equivalent ¹⁴N nuclei in the plane. It is suggested that this center, also formed by incubating the enzyme in 10% methanol followed by direct irradiation, must be the Cu_B center. This type 2 copper EPR spectrum is identical to the EPR spectrum of Cu_B reported for the isolated cytochrome bo₃ complex from Escherichia coli; and to the EPR spectrum reported for the sulfobetaine 12 heat-treated cytochrome c oxidase complex. It is argued that a small perturbation in the system causes decoupling of the magnetic coupling of the heme a₃-Cu_B binuclear center and the appearance of the type 2 EPR signal.