Reduced oxy intermediate observed in D251N cytochrome P450cam
D E Benson1, K S Suslick, S G Sligar
1Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
Biochemistry
|April 29, 1997
Summary
Researchers studied a mutant cytochrome P450cam enzyme. This mutant allows observation of a novel intermediate with an intact dioxygen bond during its catalytic cycle.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Cytochrome P450s are essential heme proteins catalyzing oxidative metabolism.
- The native cytochrome P450cam's catalytic cycle is limited by dioxygen complex reduction, preventing intermediate detection.
- A site-specific mutation (D251N) alters proton transfer, shifting the rate-determining step.
Purpose of the Study:
- To investigate the catalytic cycle of the D251N cytochrome P450cam mutant.
- To identify and characterize catalytic intermediates previously undetectable in native enzymes.
Main Methods:
- Site-directed mutagenesis to create D251N cytochrome P450cam.
- UV-visible spectroscopy to monitor spectral changes during catalytic turnover.
- Enzyme kinetics to determine rate-limiting steps.
Main Results:
- The D251N mutation shifted the rate-determining step to occur after oxy-P450 reduction.
- A new intermediate, one electron reduced from oxy-P450 with an intact O-O bond, was observed spectroscopically.
- This intermediate is stable enough for detection due to the altered catalytic pathway.
Conclusions:
- The D251N mutation facilitates the observation of a key catalytic intermediate in cytochrome P450cam.
- This finding provides new insights into the mechanism of oxygen activation and reduction in P450 enzymes.
- The study demonstrates the utility of enzyme engineering for mechanistic investigations.
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