2 CYSTEINES, 2 HISTIDINES, AND ONE METHIONINE ARE LIGANDS OF A BINUCLEAR PURPLE COPPER CENTER

Cytochrome c oxidase contains a copper center, Cu(A), which is involve d in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is compri sed Of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuratio n. Here we use a model of Cu(A), engineered into a structurally relate d but initially copperless protein, to study the structure of this cop per center. The results from biochemical analysis, site-directed mutag enesis, and electrospray mass spectrometry support the binuclear model . Two cysteines, two histidines, and one methionine are the major liga nds of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum . In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor pert urbation of the optical spectrum.