An artificial electron donor supported catalytic cycle of Pseudomonas putida cytochrome P450cam.
Putidaredoxin (PdX), the physiological effector of cytochrome P450cam (P450cam), serves to gate electron transfer into oxy-P450cam during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450cam turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450cam. We demonstrate that the catalytic cycle of wild-type P450cam can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450cam, we observed an intermediate with spectral features characteristic of compound I. The rate constant for the formation of compound I in the presence of ferrocyanide supported reaction cycle was found to be comparable to the ones observed for H2O2 supported compound I formation in wild-type P450cam, but was much lower than those observed for classical peroxidases. The results presented in this paper form the first kinetic analysis of this intermediate for an artificial electron-driven P450cam catalytic pathway in solution.
Prasad
Swati
S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India. swati@scripps.edu
Murugan
Rajamanickam
R
Mitra
Samaresh
S
D001671:Biochemistry / Q000379:methods; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003580:Cytochromes; D004579:Electron Transport; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D005295:Ferrocyanides / Q000737:chemistry / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D015233:Models, Statistical; D010544:Peroxidases / Q000378:metabolism; D011522:Protons; D016958:Pseudomonas putida / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D003580:Cytochromes; D005295:Ferrocyanides; D007477:Ions; D046911:Macromolecular Substances; D011522:Protons; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; D019475:Camphor 5-Monooxygenase; C020354:hexacyanoferrate II