Kinetic Stability of the Peroxidase Activity of Unfolded Cytochrome <i>c</i>:  Heme Degradation and Catalyst Inactivation by Hydrogen Peroxide

Unfolding converts <i>Paracoccus versutus</i> cytochrome <i>c</i>-550 into a potent peroxidase (Diederix, R. E. M.; Ubbink, M.; Canters, G. W. <i>ChemBioChem </i><b>2002</b>, <i>3,</i> 110−112). The catalytic activity is accompanied by peroxide-driven inactivation that is prevented, in part, by reducing substrate. Here, the kinetics of inactivation are described, and evidence is presented for the occurrence of a labile intermediate on the catalytic peroxidase pathway of unfolded cytochrome <i>c</i>-550. This intermediate represents a branching point, whereby the protein proceeds along either the productive pathway or self-inactivates. Reducing substrate suppresses inactivation by decreasing the steady-state concentration of the labile intermediate. Inactivation is accompanied by heme degradation. Its chemical reactivity, UV−vis, and EPR properties identify the first intermediate as hydroxyheme-cytochrome <i>c</i>-550, i.e. with heme hydroxylated at one of the heme meso positions. The occurrence of this species argues for the peroxo-iron species in the peroxidase mechanism as the labile intermediate leading to inactivated cytochrome <i>c</i>-550.