The EPR spectrum of copper in cytochrome c oxidase (EC 126.96.36.199) has been studied between 5 and 220°K, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx - 1.99, gy = 2.03, gz = 2.185, ∥ Ax(Cu) ∥ = 0.0020cm-1, ∥ Ay(Cu) ∥ = 0.0025cm-1, ∥ Az(Cu) ∥ = 0.0030cm-1. The principal values of the g tensor and the small value of ∥ Az(Cu) ∥ are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distortion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values ΔHx = 45G, ΔHy = 65G, ΔHz = 85G. A weak dipolar interaction with a low-spin ferric species about 7 Å distant is proposed to account for this anisotropy. The proposed site and stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time have been observed near g = 14and g = 3 at 5°K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.
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