An artificial electron donor supported catalytic cycle of Pseudomonas putida cytochrome P450{sub cam}

Putidaredoxin (PdX), the physiological effector of cytochrome P450{sub cam} (P450{sub cam}), serves to gate electron transfer into oxy-P450{sub cam} during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450{sub cam} turnover reaction. PdX i...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2005-09, Vol.335 (2)
Main Authors: Prasad, Swati, Murugan, Rajamanickam, Mitra, Samaresh
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
BINDING ENERGY
ELECTRON TRANSFER
ELECTRONS
FERROCYANIDES
HYDROGEN PEROXIDE
PEROXIDASES
POTASSIUM
PSEUDOMONAS
VALENCE
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Summary:Putidaredoxin (PdX), the physiological effector of cytochrome P450{sub cam} (P450{sub cam}), serves to gate electron transfer into oxy-P450{sub cam} during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450{sub cam} turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450{sub cam}. We demonstrate that the catalytic cycle of wild-type P450{sub cam} can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450{sub cam}, 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 H{sub 2}O{sub 2} supported compound I formation in wild-type P450{sub cam}, 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 P450{sub cam} catalytic pathway in solution.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2005.07.118