ic0343861_si_001.pdf (150.43 kB)
Kinetic Stability of the Peroxidase Activity of Unfolded Cytochrome c: Heme Degradation and Catalyst Inactivation by Hydrogen Peroxide
journal contribution
posted on 2003-10-04, 00:00 authored by Rutger E. M. Diederix, Maria Fittipaldi, Jonathan A. R. Worrall, Martina Huber, Marcellus Ubbink, Gerard W. CantersUnfolding converts Paracoccus versutus cytochrome c-550 into a potent peroxidase (Diederix, R. E. M.; Ubbink,
M.; Canters, G. W. ChemBioChem 2002, 3, 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 c-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 c-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 c-550.