The oxidation of o-aminophenols by cytochrome c and cytochrome oxidase. I. Enzymatic oxidations and binding of oxidation products to bovine serum albumin.

The mechanism by which a chemical agent binds to proteins is of considerable interest, particularly since a large number of compounds with carcinogenic activity have been found to bind firmly to cellular proteins at the site of tumor formation. Included in this category are the polycyclic aromatic hydrocarbons (l), the aminoazo dyes (2), N-(2.fluorenyl)acetamide (3), and the nitrogen and the sulfur mustards (4). With the exception of the mustards, binding of these agents appears to depend on prior metabolism to as yet ill-defined metabolites. In the case of AFAr the magnitude of binding to rat liver proteins in vitro paralleled the relative magnitude of hydroxylation (5). Since liver homogenates fortified with the cofactors required for hydroxylation gave an appreciable value of proteinbound radioactivity when incubated with carbon 14-labeled AFA, whereas only low values were detectable with unfortified homogenates, it has been suggested that hydroxylation is prerequisite to protein binding (6). The protein-bound radioactivity was not removable by drastic means such as solution of the protein in dilute sodium hydroxide followed by reprecipitation with acid, so that the binding referred to here is probably due to strong covalent linkages. A likely reaction path leading ultimately to covalently bound carcinogen-protein derivatives is through the intermediate formation of a quinoneimine or an N-acyl quinoneimine (quinoneimide) (5). This pathway, which requires a further oxidation of the hydroxylated metabolites, is plausible from a consideration of the structures and reactivities of the oxidized intermediates, but heretofore has lacked direct experimental support. The validity of this mechanism therefore required experimental proof that such quinonoid intermediates are indeed generated enzymatically from the hydroxylated metabolites, and that these intermediates in turn bind to proteins. The present report provides evidence that the cytochrome c cytochrome oxidase system might perform these reactions in the cell, and that these oxidized products bind readily to bovine serum albumin in a model system in vitro.