The buried charge of Asp-235 in cytochrome c peroxidase (CCP) forms an important hydrogen bond to the histidine ligand of the heme iron. The Asp-His-metal interaction, which is similar to the catalytic triad of serine proteases, is found at the active site of many metalloenzymes and is believed to modulate the character of histidine as a metal ligand. We have examined the influence of this interaction in CCP on the function, redox properties, and iron zero-field splitting in the native ferric state and its effect on the Trp-191 free radical site in the oxidized ES complex. Unlike D235A and D235N, the mutation D235E introduces very little perturbation in the X-ray crystal structure of the enzyme active site, with only minor changes in the geometry of the carboxylate-histidine interaction and no observable change at the Trp-191 free radical site. More significant effects are observed in the position of the helix containing residue Glu-235. However, the small change in hydrogen bond geometry is all that is necessary to (1) increase the reduction potential by 70 mV, (2) alter the anisotropy of the Trp-191 free radical EPR, (3) affect the activity and spin-state equilibrium, and (4) reduce the strength of the iron ligand field as measured by the zero-field splitting. The changes in the redox potential with substitution are correlated with the observed zero-field splitting, suggesting that redox control is exerted through the heme ligand by a combination of electrostatic and ligand field effects. The replacement of Asp-235 with Glu appears to result in a significantly weaker hydrogen bond in which the proton resides essentially with His-175. This hydrogen bond is nevertheless strong enough to prevent the reorientation of Trp-191 and the conversion to one of two low-spin states observed for D235A and D235N. The Asp-His-Fe interaction is therefore as important in defining the redox properties and imidazolate character of His-175 as has been proposed, yet its most important role in peroxidase function may be to correctly orient Trp-191 for efficient coupling of the free radical to the heme and to maintain a high-spin 5-coordinate heme center.
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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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