Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Gregory M. Raner, Associate Professor and Graduate Director (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
Abstract: The reaction of hydrogen peroxide and certain aromatic aldehydes with cytochrome P450BM3-F87G results in the covalent modification of the heme cofactor of this monooxygenase. Analysis of the resulting heme by electronic absorption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P4502B4, which apparently occurs via a peroxyhemiacetal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was observed that replacement of the Phe-87 in the P450BM3 by the smaller glycyl residue was essential for the modification to proceed, as the wild-type enzyme showed no spectral changes under identical conditions. The kinetics of this reaction were examined by stopped-flow spectrophotometry with 3-phenylpropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the process of heme modification was biphasic, with initial bleaching of the Soret absorbance, followed by an increase in absorbance centered at 430 nm, consistent with meso-heme adduct formation. The intermediate formed during phase I also showed an increased absorbance between 700 and 900 nm, relative to the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for phase II. All of these observations are consistent with a mechanism involving radical attack at the γ-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of which produces the γ-meso-alkyl heme derivative.
Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes
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Additional Information
- Publication
- The Journal of Inorganic Biochemistry 81: 153-160 (2000)
- Language: English
- Date: 2000
- Keywords
- Cytochrome P450, Aldehydes, Heme, Isoporphyrin, Kinetics