Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX

Abstract

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.

Aminoglutethimide-Induced Protein Free Radical Formation on Myeloperoxidase: A Potential Mechanism of Agranulocytosis

“…ABAH (42) and azide (43) are both inhibitors of MPO, and both attenuated DMPO-MPO adduct formation. Ascorbate is a substrate and also a reductant in that MPO/H 2 O 2 can oxidize ascorbate, but, perhaps more importantly, ascorbate can rapidly reduce many radical intermediates formed by peroxidases (44).…”

Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione

“…Previous studies have shown that GSH is oxidized to the glutathiyl radical by prostaglandin H synthase and that the glutathiyl radical subsequently attacks the double bond of styrene to form a carbon-centered radical trapped by DMPO (53,78). In addition, the mechanisms for addition of azide and cyanide to the heme of horseradish peroxidase have been proposed to be mediated by azidyl (79) and cyanyl (80) radicals, respectively. In this work, we detected LPO radical-derived DMPO nitrone adducts by both ELISA and Western blotting and the glutathiyl radical by ESR in the interaction of LPO with GSH.…”

RuIII(edta) mediated oxidation of azide in the presence of hydrogen peroxide. Azide versus peroxide activation

“…The above pathways involving substrate activation can be advantageous with regard to catalyst deactivation by substrate binding as reported for certain peroxidases. 23 It is noteworthy here that like in the present case, the 1 The kinetic barrier may be associated with the complex nature of the oxo-transfer/oxo-insertion process.…”