A cytochrome c variant resistant to heme degradation by hydrogen peroxide

• Published in: Chemistry & biology Vol. 7; no. 4; pp. 237 - 244
• Main Authors: Villegas, Jose A, Mauk, A Grant, Vazquez-Duhalt, Rafael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 01.04.2000
• Subjects: Biocatalysis; Cytochrome c; Cytochrome c Group - antagonists & inhibitors; Cytochrome c Group - genetics; Cytochromes c; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors - pharmacology; Enzyme Stability; heme; Heme - metabolism; Hydrogen Peroxide - pharmacology; Inactivation mechanism; Kinetics; Mutagenesis, Site-Directed; Peroxidase activity; Saccharomyces cerevisiae Proteins; Spectrophotometry; Stability; Yeasts; Cytochrome c; Biocatalysis; Stability; Peroxidase activity; Inactivation mechanism
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/S1074-5521(00)00098-3

Background: Cytochrome c has peroxidase-like activity and can catalyze the oxidation of a variety of organic substrates, including aromatic, organosulfur and lipid compounds. Like peroxidases, cytochrome c is inactivated by hydrogen peroxide. During this inactivation the heme prosthetic group is destroyed. Results: Variants of the iso-1-cytochrome c were constructed by site-directed mutagenesis and were found to be more stable in the presence of hydrogen peroxide than the wild type. No heme destruction was detected in a triple variant (Tyr67→Phe/Asn52→Ile/Cys102→Thr) with the catalytic hydrogen peroxide concentration of 1 mM, even following the loss of catalytic activity, whereas both double variants Tyr67→Phe/Cys102→Thr and Asn52→Ile/Cys102→Thr showed a greater rate of peroxide-induced heme destruction than observed with the wild-type protein. Conclusions: Heme destruction and catalytic inactivation are two independent processes. An internal water molecule (Wat166) is shown to be important in the heme destruction process. The absence of a protein radical in the resistant variant suggests that the protein radical is necessary in the heme destruction process, but presumably is not involved in the reactions leading up to the protein inactivation.