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) Biochemistry27, 5470–5476] and the peroxidase from the coprophilic fungus Coprinus macrorhizus[DePillis and Ortiz de Montellano (1989) Biochemistry28, 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 chrysosporiumare 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. macrorhizussubstantial 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 ( t 1/2= 6.3 × 10 3s) 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.