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Catalysis by Microsomal Cytochrome P450 2B4 Proceeds via a “Stable Hydroperoxo” Intermediate Identified by Freeze Quench EPR
Author(s) -
Waskell Lucy,
Pearl Naw May,
Im SangChoul,
Kunz Ryan,
Ragsdale Stephen W.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.512.8
Subject(s) - chemistry , catalysis , heterolysis , cytochrome p450 , heme , electron paramagnetic resonance , photochemistry , catalytic cycle , oxidizing agent , bond cleavage , ferric , stereochemistry , enzyme , organic chemistry , nuclear magnetic resonance , physics
Under single turnover conditions, catalysis by cytochrome P450 2B4 (P450) is 10 to 100‐fold faster in the presence of cytochrome b 5 ( b 5 ) than in the presence of P450 reductase (CPR). Both redox partners reduce oxyferrous P450 at the same rate, indicating that a step downstream of reduction accounts for the faster rate by b 5 . We hypothesized that catalysis is faster in the presence of b 5 because it causes an allosteric change in the distal heme pocket that facilitates delivery of the second proton to the hydroperoxo (Fe +3 OOH) − intermediate. This results in heterolytic O 2 bond cleavage and formation of the active oxidizing species. Conversely, we postulated that catalysis in the presence of CPR is slower because the second proton is delivered more slowly to the hydroperoxo intermediate. This predicts that the hydroperoxo intermediate will accumulate in P450 when the oxyferrous enzyme is reduced by CPR. To test this hypothesis, a prereduced P450‐CPR complex was rapidly mixed with O 2 saturated buffer. The EPR spectra of the rapidly freeze‐quenched samples demonstrated an end‐on ferric hydroperoxo intermediate, with g values of 2.32, 2.20, and 1.948, which is consistent with g values observed with hydroperoxo species of P450 camphor and P450 2B4. In summary, catalysis by P450 in the presence of CPR but not b 5 proceeds via a stable hydroperoxo intermediate under ambient conditions.