Ligand chase studies as a probe of binuclear core structure in bovine heart cytochrome c oxidase
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Exogenous ligands have been used to probe the catalytic site structure of cytochrome c oxidase. Hydrogen peroxide and formate were used separately and together to probe the binuclear center. The ligand chase study (involving both ligands) was designed with two objectives: a) to probe the binding site of formate, and b) to determine if die peroxide derivatives, 606 (P) and 580 (F) forms are structurally similar or different. Formate binding to cytochrome c oxidase resulted in the slow conformer (k^ < 0.0002 s'1) as detected by cyanide. It was previously suggested that formate is bound to the heme a3-CuB bridging site in this slow form. Ligand chase studies reported in the current work (hydrogen peroxide binding to enzyme preequilibrated with formate) suggest that formate is bound only to CuB. Hydrogen peroxide binding to 100% rapid enzyme results in sequential formation of the 606 (P) and 580 (F) species. Controversy exists as to the structural similarity or difference between these two species. Ligand chase results (formate binding to enzyme preequilibrated with hydrogen peroxide) have led to the proposal that these two forms are structurally similar.