Cytochrome c peroxidase (CcP) reacts with peroxide to form compound I, an intermediate that has an oxy-ferryl iron center and a stable indolyl radical at Trp 191. During the normal catalytic cycle, the oxy-ferryl heme and the Trp 191 radical are reduced by sequential electron transfers from ferrous cytochrome c (Cc). To investigate the role of protein structure in these electron transfer reactions, mutagenesis was used to replace Trp 191 with Phe. The Trp 191-->Phe enzyme [CcP(MI,F191)] reacts with peroxide to form an oxy-ferryl iron center and a transient porphyrin radical. The reaction of Cc from horse and yeast with peroxide-oxidized CcP(MI,F191) was characterized under transient and steady-state conditions. The rate of ET from Cc to the oxy-ferryl heme of CcP(MI,F191) was decreased by at least 10,000-fold relative to the CcP(MI) parent. This effect was observed at 20 and 100 mM ionic strength, with both yeast and horse cytochrome c as the substrate. Thus, Trp 191 is a critical component of all pathways that permit rapid reduction of the oxy-ferryl heme by Cc under these conditions. The reaction of the porphyrin radical with Cc was difficult to characterize, owing to the short half-life of this intermediate. The oxidation of Cc by this intermediate had a maximum rate constant of 32 s-1 at pH 6.0, 25 degrees C. Circumstantial evidence suggests that the porphyrin radical is not directly reduced by Cc, but is instead reduced via a protein-based radical intermediate. The steady-state activity of the mutant enzyme was 300-600-fold lower than the CcP(MI) parent, but kcat is 7-20 times greater than the rate constant for reduction of the oxy-ferryl heme under all conditions examined. Thus, the oxy-ferryl heme is not reduced to the ferric state under steady-state conditions. Transient changes in the absorption spectrum further indicate that steady-state oxidation of Cc2+ by CcP(MI,F191) occurs via reaction of peroxide with the oxy-ferryl enzyme.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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