The mechanism by which azide ion is oxidized by catalase has been the subject of considerable scrutiny for over fifty years. More recently, the oxidation of cyanamide (a structurally similar substrate) by catalase has received increased attention because of its physiological and therapeutic effects as an alcohol deterrent. Cyanamide is also a potent metabolite of thiourea, a goitrogen which blocks thyroxine biosynthesis. Oxidation of azide and cyanamide are of fundamental importance because nitric oxide and/or nitroxyl ion are products. This is of interest because of discoveries over the last decade which have documented the important physiological and pharmacological roles of nitric oxide. In the case of cyanamide oxidation by catalase, evidence has been presented that suggests that nitroxyl ion production may be important in causing the physiological and pharmacological effects observed. Despite considerable study, many of the details of cyanamide and azide oxidation by catalase remain obscure. The role of molecular oxygen is of particular interest and is not well understood. Elucidation of these details are crucial for a fundamental understanding of this enzyme. Thus, the overall objective of this research is to further elucidate mechanistic details of azide and cyanamide oxidation as they occur in bovine liver catalase. Specific objectives are as follows: 1) To determine the mechanism by which molecular oxygen consumption occurs during catalase mediated oxidation of azide and cyanamide; 2) To determine if both nitric oxide and nitroxyl ion are generated during catalase mediated oxidation of azide and cyanamide; 3) To determine the origin of the oxygen atom in nitric oxide and nitroxyl ion that is produced during catalase mediated oxidation of azide and cyanamide (i.e., does the oxygen atom come from molecular oxygen or hydrogen peroxide?); 4) To characterize catalase mediated cyanamide oxidation. Unlike azide, oxidation of cyanamide has not been studied in detail. Specifically, we will determine the kinetic constants (Km, kcat, Vmax, etc.) associated with this reaction, its pH and temperature optima and other parameters. Most importantly we will determine if, as is the case with azide, the reaction mechanism involves formation of an intermediary catalase ferrous-NO complex.
