X-RAY-ABSORPTION STUDIES ON THE MIXED-VALENCE AND FULLY REDUCED FORMSOF THE SOLUBLE CU-A DOMAINS OF CYTOCHROME-C-OXIDASE

Cytochrome oxidase is the terminal oxidase in both prokaryotic and euk aryotic cells and is responsible for the generation of cellular energy via the process known as oxidative phosphorylation: The enzyme contai ns two Fe and three Cu centers which together provide the redox machin ery for the reduction of O-2 to water. Recently, X-ray crystallography has provided the first three-dimensional description of the coordinat ion spheres of the metal centers: However, the structures show the met al sites at low resolution, and in order to fully understand the mecha nism of the reaction, it is desirable to determine the metrical detail s (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 ce nter, 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 similar to 2.4 Angstrom. X-ray absorption spectroscopy, w hich 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 thermophi lus and Bacillus subtilis. The results have defined the structure of t he Cu-A core as a Cu2S2 diamond with Cu-S bond lengths of 2.3 Angstrom , Cu-Cu = 2.44 Angstrom, and very acute Cu-S-Cu angles of 65 degrees. One-electron reduction produces only minor changes in the core geometr y, with the Cu-S and Cu-Cu bond lengths increasing to 2.33 and 2.51 An gstrom, respectively, but with the Cu-S-Cu angle remaining unchanged a t 65 degrees. The unusually high Cu-S Debye-Waller terms imply that th ere is significant asymmetry in the Cu2S2 diamond core derived from in equivalent Cu-S bond lengths. Both the metrical parameters and the tem perature dependence of the Debye-Waller factors exhibit subtle differe nces between the mixed-valence and fully reduced proteins which sugges t 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 occu rring during electron transfer. Thus, they provide an excellent ration alization for the very low reorganizational energy, lambda, observed f or the Cu-A center.