Further studies on the inactivation by sodium azide of lignin peroxidase from Phanerochaete chrysosporium.
Azide ion is a mechanism-based inactivator of horseradish peroxidase [Ortiz de Montellano et al. (1988) Biochemistry 27, 5470-5476] and the peroxidase from the coprophilic fungus Coprinus macrorhizus [DePillis and Ortiz de Montellano (1989) Biochemistry 28, 7947-7952]. These peroxidases mediate the one-electron oxidation of azide ion-forming azidyl radical. Inactivation of these enzymes is caused by covalent modification of the heme prosthetic groups by azidyl radical. Lignin peroxidases from the wood-rotting fungus Phanerochaete chrysosporium are also inactivated when they catalyze oxidation of azide ion [Tuisel et al. (1991) Arch. Biochem. Biophys. 288, 456-462; DePillis et al. (1990) Arch. Biochem. Biophys. 280, 217-223]. Following inactivation of horseradish peroxidase and the peroxidase from C. macrorhizus substantial amounts of azidyl-heme adducts have been found. Only trace amounts of such adducts have been found following azide-mediated inactivation of lignin peroxidase. Nevertheless, we have shown that during oxidation of azide by lignin peroxidase H8 destruction of heme occurred and a substantial fraction of the enzyme is irreversibly inactivated. However, the rest of the enzyme forms a relatively stable ferrous-nitric oxide (NO) complex. Although this complex appears to be an inactivated form of the enzyme, we have shown that, when present as the ferrous-NO complex, the enzyme is actually protected from inactivation. The lignin peroxidase ferrous-NO complex reverts slowly (t1/2 = 6.3 x 10(3) s) to the ferric form. Reversion is accelerated if the complex is chromatographed on a PD-10 (Sephadex G-25) column or if veratryl alcohol is added. If azide and hydrogen peroxide (a required cosubstrate) are present (or added), the enzyme undergoes another cycle of catalysis and further inactivation. A detailed reaction mechanism is proposed that is consistent with our experimental observations, the chemistry of azide, and our current understanding of peroxidases.
Tatarko
M
M
Department of Biological Sciences, University of Notre Dame, Indiana 46556, USA.
Bumpus
J A
JA
D001386:Azides / Q000737:chemistry / Q000494:pharmacology*; D001487:Basidiomycota / Q000201:enzymology*; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D009609:Nitrous Oxide / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013057:Spectrum Analysis
D001386:Azides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006418:Heme; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase; D009609:Nitrous Oxide