Kinetic characterization of compound I formation in the thermostable cytochrome P450 CYP119.
The kinetics of formation and breakdown of the putative active oxygenating intermediate in cytochrome P450, a ferryl-oxo-(pi) porphyrin cation radical (Compound I), have been analyzed in the reaction of a thermostable P450, CYP119, with meta-chloroperoxybenzoic acid (m-CPBA). Upon rapid mixing of m-CPBA with the ferric form of CYP119, an intermediate with spectral features characteristic of a ferryl-oxo-(pi) porphyrin cation radical was clearly observed and identified by the absorption maxima at 370, 610, and 690 nm. The rate constant for the formation of Compound I was 3.20 (+/-0.3) x 10(5) m(-1) s(-1) at pH 7.0, 4 degrees C, and this rate decreased with increasing pH. Compound I of CYP119 decomposed back to the ferric form with a first order rate constant of 29.4 +/- 3.4 s(-1), which increased with increasing pH. These findings form the first kinetic analysis of Compound I formation and decay in the reaction of m-CPBA with ferric P450.
Kellner
David G
DG
Departments of Biochemistry, Chemistry, and the College of Medicine, University of Illinois, Urbana, Illinois 61801, USA.
Hung
Shao-Ching
SC
Weiss
Kara E
KE
Sligar
Stephen G
SG
D019843:Archaeal Proteins; D002723:Chlorobenzoates / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D016877:Oxidants / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D011166:Porphyrins / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors
D019843:Archaeal Proteins; D002723:Chlorobenzoates; D007202:Indicators and Reagents; D016877:Oxidants; D011166:Porphyrins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus; C000433:3-chloroperbenzoic acid