Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.
Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.
DePillis
G D
GD
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
Sishta
B P
BP
Mauk
A G
AG
Ortiz de Montellano
P R
PR
D001386:Azides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005295:Ferrocyanides / Q000378:metabolism; D006139:Guaiacol / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000494:pharmacology
D001386:Azides; D003574:Cytochrome c Group; D005295:Ferrocyanides; D006420:Hemeproteins; D010659:Phenylhydrazines; C030299:phenylhydrazine; D006139:Guaiacol; D003578:Cytochrome-c Peroxidase; C020354:hexacyanoferrate II