Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes.
Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.
Jiang
Yongying
Y
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, California 94143-2280, USA.
He
Xiang
X
Ortiz de Montellano
Paul R
PR
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D000081008:Bicyclic Monoterpenes; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2 / Q000502:physiology; D019389:Cytochrome P-450 CYP2D6 / Q000502:physiology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017666:Deuterium Oxide / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007536:Isomerism; D007930:Leucine / Q000235:genetics; D039821:Monoterpenes / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction
D001426:Bacterial Proteins; D000081008:Bicyclic Monoterpenes; D005609:Free Radicals; D039821:Monoterpenes; C005790:beta-thujone; D003577:Cytochrome P-450 Enzyme System; C504573:CYP1A2 protein, human; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D019475:Camphor 5-Monooxygenase; D007930:Leucine; D017666:Deuterium Oxide; D000409:Alanine