TY - JOUR
T1 - Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate
AU - Cooper, Harriet L.R.
AU - Groves, John Taylor
N1 - Funding Information:
We are grateful for support of this research by the National Institutes of Health (2R37 GM036298).
PY - 2011/3/1
Y1 - 2011/3/1
N2 - The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1. The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol, was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios of rearranged and unrearranged products to be 120, 13 and 1 ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9 × 1010 s-1, 7.2 × 1010 s -1 and 1.0 × 1012 s-1. For the model iron porphyrin, FeTMPyP, a radical lifetime of 81 ps and a rebound rate of 1.2 × 1010 s-1 were determined. These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron porphyrin suggest that for these systems a considerable amount of the intermediate carbon radical is trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon radical in [FeIV-OH -R] can compete with cage collapse to form a C-O bond. The results could be significant with regard to our understanding of iron-catalyzed C-H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1.
AB - The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1. The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol, was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios of rearranged and unrearranged products to be 120, 13 and 1 ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9 × 1010 s-1, 7.2 × 1010 s -1 and 1.0 × 1012 s-1. For the model iron porphyrin, FeTMPyP, a radical lifetime of 81 ps and a rebound rate of 1.2 × 1010 s-1 were determined. These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron porphyrin suggest that for these systems a considerable amount of the intermediate carbon radical is trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon radical in [FeIV-OH -R] can compete with cage collapse to form a C-O bond. The results could be significant with regard to our understanding of iron-catalyzed C-H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1.
KW - Hydroxylation
KW - Mechanism
KW - Oxidation
KW - Radical
KW - Rebound
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U2 - 10.1016/j.abb.2010.11.001
DO - 10.1016/j.abb.2010.11.001
M3 - Article
C2 - 21075070
AN - SCOPUS:79951572088
SN - 0003-9861
VL - 507
SP - 111
EP - 118
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -