Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450(cam).
The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450(cam) (P450(cam)) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H(2)O(2) and m-CPBA with substrate-bound forms of P450(cam), we observed an intermediate with spectral features characteristic of compound I. Unlike in H(2)O(2), kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450(cam) and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450(cam).
Prasad
Swati
S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, 400005 Mumbai, India. prasad@biochem.wustl.edu
Mitra
Samaresh
S
D019475:Camphor 5-Monooxygenase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D015233:Models, Statistical; D010100:Oxygen / Q000378:metabolism; D010545:Peroxides / Q000737:chemistry; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D010545:Peroxides; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D019475:Camphor 5-Monooxygenase; D010100:Oxygen