The reactions of azide with catalase and their significance

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Abstract
The rates and stoicheiometries of the reactions of mammalian catalase with azide and peroxide have been studied. Azide diminishes the rate of compound II formation in the glucose oxidase-glucose-catalase system, reacting with compound I as a hydrogen donor like alcohols. The final product of the reaction with azide and peroxide in air or nitorgen is nitric oxide-ferrocatalase; under carbon monoxide, the product is carbon monoxide-ferrocatalase. Similar products are formed whether (a) hydrogen or ethyl hydroperoxide is used to form compound I, (b) azide or hydroxylamine is present as hydrogen donor, (c) liver or blood catalase is used. The spectra of the nitric oxide and carbon monoxide derivatives of the ferrous form of a reducible catalase are closely similar to those produced indirectly. The rates of formation of carbon monoxide-ferrocatalase and nitric oxide-ferro-catalase under anaerobic conditions are proportional to peroxide concentration, indicating compound I formation to be the rate-limiting step; but the rate of formation of nitric oxide-ferrocatalase is only one-tenth that of carbon monoxide-ferrocatalase formation. The formation of nitric oxide-ferrocatalase in air is a diphasic reaction. Nitric oxide-ferrocatalase is vulnerable to oxidation by oxygen, but carbon monoxide-ferrocatalase is stable; however, carbon monoxide-ferrocatalase is dissociated by light, the first detectable product being ferric catalase. One molar equivalent of hydrogen peroxide is required to form one equivalent of carbon monoxide-ferrocatalase in the presence of an excess of azide; five to twelve molar equivalents of hydrogen peroxide are required to form one equivalent of nitric oxide-ferrocatalase, the precise ratio depending on the state of aerobiosis and the catalase sample used. Hydroxylamine and hydrazine also give rise to nitric oxide-ferrocatalase in the presence of peroxide, but in neither case is the reaction simpler than that with azide. Horse-radish peroxidase compound I reacts with azide to give a firmly bound ferric derivative, reducible to a second ferric derivative from which peroxidase may be regenerated. The reactions of catalase are interpreted in terms of two reactions of compound I with azide, one (80-90% of the total turnover) giving ferrocatalase, nitrous oxide and a nitrogen (N0) radical the other (10-20% of the total turnover) giving nitric oxide-ferrocatalase and nitrogen. These correspond to reactions suggested by Theorell and Ehrenberg (1952b), and by Foulkes and Lemberg (1949) respectively. The reactions with azide support the view that the oxidation of "multi-electron" donors by catalase compound I involves the formation of a complex between the donor molecule and the haematin iron. A structure for compound I is put forward in which the peroxide moiety is associated with the porphyrin ring, and some consequences for reaction mechanisms are examined in the light of this hypothesis.