Catalases reaction with azide

Reactions of hemoglobin with cyanide and the magnetic properties of the cyanide complex. Equilibrium constant of the reaction. Reaction of cyanide with catalase and peroxidase. Magnetic properties of products. Reaction of hemoglobin with fluoride and its relation to the reaction with hydroxyl ion. Equilibrium constants for reactions of fluoride with cytochrome c, catalase, and peroxidase. Magnetic effects associated with these reactions. Velocity of reaction of ferrihemoglobin with hydro-sulfide. Magnetic properties of product. Reactions with azide. Reactions of hydroxyl ion with various heme proteins. Reconsideration of all the results in connection with possibility of interactions between hemes. 

Frasch (45, 46) has shown that the OEC can catalyze an azide-insensitive catalase reaction in the dark. The activity can be directly associated with the OEC because (1) competitive inhibitors of water oxidation are also competitive inhibitors of the catalase activity and (2) the K for water oxidation and catalase activity are essentially identical. The enzyme apparently cycles in this case between S0 and S2. Mano and co-workers (47) showed that the Si/S i states are also competent to carry out catalase reactions however, this reaction is highly pH-depen-dent. For example, at pH 8.8, the Si state can oxidize H202 to 02, but S i is incapable of completing the reaction cycle however, if the pH is lowered to pH 6 steady-state measurements of oxygen evolution can be gathered. Just as is the case with water oxidation, these catalase reactions... 

Catalase was found to form an intermediate compound in the presence of hydrogen peroxide (Chance, 69). The spectrum was measured from 380-430 nqi and is slightly shifted toward the visible as compared with free catalase. The complex shows no similarities to cyan-catalase or the compound formed when peroxide is added to azide catalase. Its formation is very rapid, the bimolecular velocity constant having a value of about 3 X 107 M.-1 sec.-1. In the absence of added hydrogen donors, the complex decomposes slowly according to a first order reaction with a velocity constant of about 0.02 sec.-1. This catalase complex thus resembles the green primary hydrogen peroxide complex of peroxidase. 

The inhibitors of tryptophan pyrrolase may be classified into those that prevent activation of the enzyme and those that inhibit the active form. Catalase, as mentioned earlier, was required in certain amounts to balance peroxide-generating systems, but inhibited in higher concentrations. These effects have now been shown to concern only the activation process, and catalase has been found to have no influence on the activated tryptophan pyrrolase (Tanaka and Knox, 1959). Similarly, peroxides are required for the activation, but cause irreversible inactivation if added in the absence of tryptophan. Cyanide is a potent inhibitor if added before activation, but has very little effect if added during the reaction. Ferricyanide causes almost complete cessation of activity when added during the reaction, as was expected if only the ferrous form of the enzyme were active. This interpretation is supported by the reactivation of ferricyanide-treated enzyme by ascorbic acid, which presumably reduces the iron back to the ferrous state. However, if cyanide is added before the ascorbic acid, there is very little reactivation. Carbon monoxide causes an inhibition that is reversed by light. All of these observations are consistent with a model in which an inactive ferric enzyme is reduced to an active form by peroxide and tryptophan. The ferric form combines readily with cyanide (and also with azide and hydroxylamine), while the ferrous form combines with carbon monoxide. 

The donor type D5 comprises the two species azide and hydroxy-lamine. These both react with the enz5mie in the presence of peroxide to give rise to ferrous forms of catalase, otherwise normally inaccessible (catalase is the only common hemoprotein that is nonreducible by dithionite). The final inhibited form of catalase in the presence of azide and peroxide is NO-ferrocatalase, but not every azide molecule becomes an NO only in the presence of CO is there a stoichiometric inhibition of enzyme by peroxide with formation of 1 equiv of CO-ferrocatalase for every peroxide molecule added (43). This suggested a three-electron reduction of compound I either to give ferrocatalase, N2, and NO (10-20% total) or to give ferrocatalase, N, and N2O (80-90% total). However, Kalyanaraman et al. (45) have demonstrated the formation of the azidyl (N=N=N ) radical in the reaction, and Lardinois... 

Catalase reacts reversibly with some weak acids forming spectroscopically and magnetically distinct noncovalent derivatives. Of these, catalase-cyanide, -azide, -fluoride, -formate, and -acetate complexes have been extensively studied (37, 135, 136) and reviewed in some detail (16-18). Briefly, there is a consensus that such reactions do not involve heme-heme interaction and, with the possible exception of carboxylate ligands (102), all presumably result in replacement of the proximal aquo ligand at Ls in a stoichiometric reaction shown in Eq. (11) ... 

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