On the nature of cysteine coordination to CuA in cytochrome c oxidase.

Abstract

The resolution of new features in the 1H electron nuclear double resonance (ENDOR) spectrum of the oxidized CuA site in beef heart cytochrome c oxidase is presented. In a previous study, we assigned resonances in the CuA ENDOR spectrum to hyperfine interactions of methylene protons on one or two cysteine ligands to CuA (Stevens, T.H., Martin, C.T., Wang, H., Brudvig, G.W., Scholes, C.P., and Chan, S.I. (1982) J. Biol. Chem. 257, 12106-12113). In this work, a new 1H ENDOR resonance in the beef heart CuA ENDOR spectrum is reported and can be assigned to either anisotropy in a previously resolved cysteine methylene proton hyperfine interaction (Aiso = 12 MHz, Aaniso = 2.5 MHz) or to a third isotropic hyperfine coupling (A = 13.6 MHz) to a cysteine methylene proton of a second cysteine ligand to copper. In either case, the 1H ENDOR results require the delocalization of approximately 50% of the unpaired spin from copper onto either one or two cysteine ligands to CuA. To characterize further the CuA site, we have prepared yeast cytochrome c oxidase incorporating isotopically substituted [beta-13C]cysteine. The CuA ENDOR spectrum of this species shows only one clearly resolved 13C hyperfine interaction (A = 3.6 MHz). This result confirms the assignment of at least one strongly interacting cysteine ligand to CuA and suggests that if the assignment of two cysteine ligands to CuA is correct, the two cysteines interact with copper in a highly symmetric manner. A recent extended x-ray absorption fine structure study of native and modified forms of cytochrome c oxidase indicates the coordination of two sulfur ligands to CuA (Li, P.M., Gelles, J., Chan, S.I., Sullivan R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095). In light of the new possibility of two symmetrically coordinated cysteine ligands to CuA, we propose a molecular orbital description of the oxidized CuA site which is characterized by a high degree of delocalization of unpaired spin away from copper and onto a pair of symmetrically coordinated cysteine sulfur ligands. We also present a protein model for the CuA site in which two cysteine ligands derived from subunit II lie on the face of an alpha-helix. This structure would allow the unprecedented stable coordination of two cysteine thiolate sulfurs to copper and may provide a mechanism for the redox-linked proton pumping by cytochrome c oxidase.