Infrared Spectroscopic and Mutational Studies on Putidaredoxin-Induced Conformational Changes in Ferrous CO-P450cam
| Autor: | Yoshitsugu Shiro, Yoko Kimata-Ariga, Tsuyoshi Egawa, Sam-Yong Park, Yuzuru Ishimura, Takako Hishiki, Shin-ichi Adachi, Akiko Tarumi, Hideo Shimada, Makoto Suematsu, Shingo Nagano |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
endocrine system
Conformational change Camphor 5-Monooxygenase Spectrophotometry Infrared Cytochrome Protein Conformation Stereochemistry Crystallography X-Ray Biochemistry Catalysis Hydroxylation chemistry.chemical_compound Electron transfer Bacterial Proteins Oxidoreductase Ferrous Compounds Heme chemistry.chemical_classification biology Pseudomonas putida Active site Ligand (biochemistry) Camphor chemistry Mutagenesis Site-Directed biology.protein Ferredoxins Crystallization Oxidation-Reduction |
| Zdroj: | Biochemistry. 42:14507-14514 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi035410p |
| Popis: | Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm - 1 . The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm - 1 band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg 112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O 2 and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|