Kinetic stability of the peroxidase activity of unfolded cytochrome c: heme degradation and catalyst inactivation by hydrogen peroxide.
Unfolding 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.
Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Rutger E M
RE
Diederix
Maria
M
Fittipaldi
Jonathan A R
JA
Worrall
Martina
M
Huber
Marcellus
M
Ubbink
Gerard W
GW
Canters
D002384:Catalysis; D003574:Cytochrome c Group / Q000378:metabolism*; D004795:Enzyme Stability; D006139:Guaiacol / Q000494:pharmacology; D006418:Heme / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*
D003574:Cytochrome c Group; D006418:Heme; D006139:Guaiacol; C031732:cytochrome C-550; D006861:Hydrogen Peroxide; D010544:Peroxidases