Photooxidation of cytochrome P450-BM3.
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 (RuIIK97C-FeIIIP450), in order to promote electron transfer from the Fe(III) porphyrin to photogenerated Ru(III). The 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.
Beckman Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
Maraia E
ME
Ener
Young-Tae
YT
Lee
Jay R
JR
Winkler
Harry B
HB
Gray
Lionel
L
Cheruzel
D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D008956:Models, Chemical; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D010084:Oxidation-Reduction; D055668:Photochemical Processes; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases