X-ray absorption studies on the mixed-valence and fully reduced forms of the soluble Cu(A) domains of cytochrome c oxidase

Cytochrome oxidase is the terminal oxidase in both prokaryotic and eukaryotic cells and is responsible for the generation of cellular energy via the process known as oxidative phosphorylation. The enzyme contains two Fe and three Cu centers which together provide the redox machinery for the reduction of O₂ to water. Recently, X-ray crystallography has provided the first three-dimensional description of the coordination spheres of the metal centers. However, the structures show the metal sites at low resolution, and in order to fully understand the mechanism of the reaction, it is desirable to determine the metrical details (bond lengths and angles) to much higher precision. X-ray absorption spectroscopy is unique in its ability to provide such detail, and we have applied the technique to determining the structure of the Cu(A) center, a thiolate-bridged binuclear copper cluster in which the coppers are bridged by two cysteine ligands and have an extremely short Cu- Cu distance of ~2.4 Å. X-ray absorption spectroscopy, which had previously predicted the short Cu-Cu distance, has been used to further refine the structural details of the site in both the mixed-valence and fully reduced forms of the enzymes from Thermus thermophilus and Bacillus subtilis. The results have defined the structure of the Cu(A) core as a Cu₂S₂ diamond with Cu-S bond lengths of 2.3 Å, Cu-Cu = 2.44 Å, and very acute Cu-S-Cu angles of 65°. One-electron reduction produces only minor changes in the core geometry, with the Cu-S and Cu-Cu bond lengths increasing to 2.33 and 2.51 Å, respectively, but with the Cu-S-Cu angle remaining unchanged at 65°. The unusually high Cu-S Debye-Waller terms imply that there is significant asymmetry in the Cu₂S₂ diamond core derived from inequivalent Cu-S bond lengths. Both the metrical parameters and the temperature dependence of the Debye-Waller factors exhibit subtle differences between the mixed-valence and fully reduced proteins which suggest that the short distance may be the result, in part, of a weak metal-metal bond. The results suggest that the function of the unusual CU(A) cluster is to provide a site with minimal structural perturbation occurring during electron transfer. Thus, they provide an excellent rationalization for the very low reorganizational energy, λ, observed for the Cu(A) center.