Azide catalase

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 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... 

Both classical type A enzymes (clade III) and the heme d family (clade II) show a comparatively high sensitivity to azide inhibition and are reduced to ferrous forms in the presence of peroxide and azide (47). In contrast, the catalase-peroxidase (type B) enzymes (see below) are only weakly azide-sensitive. 

Catalases bind or react with a number of molecules that can be either substrates (hydrogen peroxide and some small alcohols) or inhibitors (cyanide, azide, etc.) (see Section IV,B). Several such intermediates have... 

Crystal structures of manganese catalases (in the (111)2 oxidation state) from Lactobacillus plantarum,its azide-inhibited complex, " and from Thermus thermophilus have been determined. There are differences between the structures that may reflect distinct biological functions for the two enzymes, the L. plantarum enzyme functions only as a catalase, while the T. thermo-philus enzyme may function as a catalase/peroxidase. The active sites are conserved in the two enzymes and are shown schematically in Figure 32. Each subunit contains an Mu2 active site,... 

The antibody-dependent lysis of tumor cells by PMNs exhibited some of the characteristics of damage mediated by products of the burst in the presence of tumor cells there was increased consumption of O2, increased formation of O and activation of the hexose monosphosphate shunt However, although a reduction in the concentration of O2 in the medium inhibited lysis neither catalase nor superoxide dismutase inhibited. The lack of effect on these enzymes was attributed to their inability to interpose themselves between the plasma membranes of the PMN and its target. Similar conclusions were reached by Clark, and Klebanoff whose data incriminated the products of the burst by the reduced killing of tumor cells by PMNs from patients with chronic granulomatous disease. Myeloperoxidase, however, appeared not to he required since neither azide or cyanide inhibited and killing by PMNs from patients with inherited deficiency of myeloperoxidase was normal. 

However, when PMNs were stimulated, not by antibody on the surface of the tumor cell, but instead by Concanavalin Aor by opsonized zymosan myeloperoxidase did appear to mediate the killing azide and cyanide inhibited the killing, halides were required, catalase inhibited, and PMNs from patients with either hereditary deficiency of myeloperoxidase or chronic granulomatous disease were... 

Complex 52 is similar to Mn-catalase in that it is azide insensitive and forms a MnnMnm species on addition of hydroxylamine and a MnIIIMnIV catalyti-cally inactive form with an EPR spectrum closely resembling that of the enzyme. Importantly, the complex maintains its dinuclear structure in solution, while cycling between the MnnMnn, MnmMnm oxidation states and shows a good catalytic rate (kcat = 13 1 sec-1) and stability (>1000 turnovers). Catalases, however, are approximately 3000 times more efficient. 

Activity measurements. Photosynthetic 02 exchange was measured using an 02 electrode (Rank Brothers, Bottisham, U.K.) illuminated with saturating white light (360 pE. m 2 s ) according to Stewart and Bendall (1980) except that azide was substituted for catalase in PSI assay and 6 mM DMBQ was used as electron acceptor of PSII. 

Aksoy Y, Balk M, Ogus I et al (2004) The mechanism of inhibition of human erythrocyte catalase by azide. Turk J Biol 28 65-70... 

A variety of anions, including azide, chloride, and fluoride, are inhibitors of Mn catalase. It is difficult to define the effect of these inhibitors using EPR, because the Mn(III)-Mn(III) derivative is EPR-silent and the Mn(II)-Mn(II) derivative is only EPR-active in the presence of added anions. XANES is an ideal probe, however, because it is sensitive to all of the Mn in the system. As expected, treatment with halide alone has no effect on Mn oxidation state. However, treatment with fluoride or chloride in the presence of H202 gives complete reduction of the Mn to Mn(II) (data not shown) (23, 24). The same result is obtained regardless of whether one starts with the reduced enzyme, the autooxidized enzyme (see eq 8) or the as-isolated enzyme. This result provides direct evidence that the halides inhibit the enzyme by trapping it in the reduced valence state. 

Although manganese catalases have often been referred to as azide insensitive/ these enzymes actually are inhibited by azide and related molecules albeit at higher concentrations than are necessary for the heme enzymes. Penner-Hahn and co-workers (22) have shown that HN3 is the likely protonation state of the inhibitor and have calculated an apparent of 80 mM. Slope replots of the pH dependence of azide inhibition are linear with a slope of 1. These data can be used to calculate a true K of 300 mM. Because azide is a competitive inhibitor with respect to peroxide, it is likely that azide is bound directly to the manganese center. Recent EPR and lH paramagnetic relaxation enhancement studies support this viewpoint. Other inhibitors include fluoride and thiocyanide. All of the reported inhibition studies are consistent with the catalase cycle and hydroxylamine inhibition of the catalase cycle. 

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... 

The L. plantarum catalase was described initially as azide insensitive because the reaction is not inhibited at azide concentrations that would easily destroy heme catalase activity. However, Penner-Hahn (22) has shown that this enzyme can be inhibited by azide if the concentrations are sufficiently high. The = 80 mM (pH 7) and is pH dependent. The [Mn(III)(2-OH-(5-Cl-sal)pn)]2 is not inhibited in acetonitrile to saturating concentrations of azide ( 50 mM). 

Figure 11. MCD spectra at 4 °C of the (A) ferric azide and (B) cyanide adducts of HPII catalase (----) and of methylchlorin-reconstituted myoglobin (----). MCD spectra were obtained using a JASCO J-500A spectro-...

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