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Published November 2, 2010 | Supplemental Material + Published
Journal Article Open

Photooxidation of cytochrome P450-BM3

Abstract

High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru^(II) photosensitizer to a nonnative cysteine near the heme (Ru^(II)_(K97C)-Fe^(III)_(P450)), in order to promote electron transfer from the Fe^(III) porphyrin to photogenerated Ru^(III). The Ru^(II)_(K97C)Fe^(III)_(P450) conjugate was structurally characterized by X-ray crystallography (2.4 Å resolution; Ru-Fe distance, 24 Å). Flash-quench oxidation of the ferric-aquo heme produces an Fe^(IV)-hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.

Additional Information

© 2010 National Academy of Sciences. Contributed by Harry B. Gray, August 19, 2010 (sent for review July 2, 2010). Published online before print October 14, 2010. This research was supported by the National Institutes of Health (DK019038) and The Arnold and Mabel Beckman Foundation. L.C. thanks Dr. Phoebe Glazer for helpful discussions. Author contributions: M.E.E., J.R.W., H.B.G., and L.C. designed research; M.E.E., Y.-T.L., and L.C. performed research; M.E.E., J.R.W., and L.C. analyzed data; and M.E.E., J.R.W., H.B.G., and L.C. wrote the paper. The authors declare no conflict of interest. Data deposition: The crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3NPL and Research Collaboratory for Structural Bioinformatics (RCSB) ID code RCSB060120).

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Published - Ener2010p11943P_Natl_Acad_Sci_Usa.pdf

Supplemental Material - pnas.1012381107_SI.pdf

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August 22, 2023
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