Characterization of the microsomal cytochrome P450 2B4 O2 activation intermediates by cryoreduction and electron paramagnetic resonance.
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 >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.
Davydov
Roman
R
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Tech K148, Evanston, Illinois 60208-3113, USA.
Razeghifard
Reza
R
Im
Sang-Choul
SC
Waskell
Lucy
L
Hoffman
Brian M
BM
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics; D000072467:Cytochrome P450 Family 2; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005615:Freezing; D006427:Hemin; D008861:Microsomes / Q000201:enzymology*; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*
D006427:Hemin; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D010100:Oxygen