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The oxy-ferrous complex of cytochrome P450 2B4 (2B4) has been prepared at -40 degrees C with and without bound substrate [butylated hydroxytoluene (BHT)] and radiolytically one-electron cryoreduced at 77 K. Electron paramagnetic resonance (EPR) shows that in both cases the observed product of cryoreduction is the hydroperoxo-ferriheme species, indicating that the microsomal P450 contains an efficient distal-pocket proton-delivery network. In the absence of substrate, two distinct hydroperoxo-ferriheme signals are observed, reflecting the presence of two major conformational substates in the oxy-ferrous precursor. Only one species is observed when BHT is bound, indicating a more ordered active site. BHT binding also changes the g-tensor components of the hydroperoxo-ferric 2B4 intermediate, indicating that the substrate modulates the properties of this intermediate. Step annealing the cryoreduced ternary 2B4 complex at &gt;or=175 K causes the loss of hydroperoxo-ferric 2B4 and the parallel appearance of high-spin ferric 2B4; liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis shows that in this process BHT is quantitatively converted to two products, hydroxymethyl BHT (1) and 3-hydroxy- tert-butyl BHT (2). This implies that the hydroperoxo-ferric 2B4 prepared by cryoreduction is catalytically active and that the high-spin state observed after annealing contains an enzyme-bound product of BHT monooxygenation. The ratio of products generated during cryoreduction and annealing (6.2/1) is significantly different from the ratio (2.5/1) at ambient temperature. These findings suggest that substrate is held more rigidly relative to the oxidizing species at low temperatures and/or that dissociation of FeOOH is inhibited at low temperature. As in experiments under ambient conditions, product formation is not observed with the inactive F429H 2B4 mutant.

Characterization of the Microsomal Cytochrome P450 2B4 O2 Activation Intermediates by Cryoreduction and Electron Paramagnetic Resonance

Characterization of the Microsomal Cytochrome P450 2B4 O₂ Activation Intermediates by Cryoreduction and Electron Paramagnetic Resonance