title: Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450.
abstract: From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, D-13125 Berlin, Germany. christiane_jung@hotmail.com
authors.forename: Christiane
authors.initials: C
authors.lastname: Jung
authors.forename: Volker
authors.initials: V
authors.lastname: Schünemann
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Lendzian
authors.forename: Alfred X
authors.initials: AX
authors.lastname: Trautwein
authors.forename: Jörg
authors.initials: J
authors.lastname: Contzen
authors.forename: Marcus
authors.initials: M
authors.lastname: Galander
authors.forename: Lars H
authors.initials: LH
authors.lastname: Böttger
authors.forename: Matthias
authors.initials: M
authors.lastname: Richter
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Barra
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006418:Heme / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D014443:Tyrosine; D006418:Heme; D014364:Tryptophan; D003577:Cytochrome P-450 Enzyme System; D007501:Iron