Engineering Cytochrome c Peroxidase into Cytochrome P450: A Proximal Effect on Heme−Thiolate Ligation
| Autor: | Jeffrey A. Sigman, Yi Lu, John H. Dawson, Alycen E. Pond |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Cytochrome
Stereochemistry Mutant Cytochrome c Group Heme Cysteic acid Ligands Protein Engineering Ferric Compounds Biochemistry law.invention chemistry.chemical_compound Cytochrome P-450 Enzyme System Leucine law Escherichia coli medicine Histidine Cysteine Electron paramagnetic resonance Aspartic Acid biology Cytochrome c peroxidase Chemistry Circular Dichroism Electron Spin Resonance Spectroscopy Imidazoles Cytochromes c Cytochrome P450 Cytochrome-c Peroxidase Mutagenesis Site-Directed biology.protein Ferric Spectrophotometry Ultraviolet medicine.drug |
| Zdroj: | ResearcherID |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi990815o |
| Popis: | In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation. |
| Databáze: | OpenAIRE |
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