Stabilization and spectroscopic characterization of the dioxygen complex of wild-type cytochrome P4502B4 (CYP2B4) and its distal side E301Q, T302A and proximal side F429H mutants at subzero temperatures.
Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O₂) complex of CYP2B4, homogeneous O₂ complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~-50°C in mixed solvents (60-70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101). However, the oxyferrous complexes of the T302A and E301Q CYP2B4 mutants have significantly perturbed electronic structure (~4 nm and ~3 nm red-shifted Soret features, respectively) compared to that of the wild-type oxyferrous complex. On the other hand, the heme proximal side mutant, CYP2B4 F429H, undergoes relatively facile conversion to a partially (~50%) denatured (P420) form upon reduction. The structural changes in the heme pocket environments of the CYP2B4 mutants that lead to the spectroscopic distinctions reported herein can be related to the differences in oxidation activities of wild-type CYP2B4 and its E301Q, T302A and F429H mutants.
Perera
Roshan
R
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA.
Sono
Masanori
M
Kinloch
Ryan
R
Zhang
Haoming
H
Tarasev
Michael
M
Im
Sang-Choul
SC
Waskell
Lucy
L
Dawson
John H
JH
D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001589:Benzphetamine / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002942:Circular Dichroism; D003080:Cold Temperature; D000072467:Cytochrome P450 Family 2; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013053:Spectrophotometry; D013379:Substrate Specificity
D009942:Organometallic Compounds; D001589:Benzphetamine; D006418:Heme; D007501:Iron; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D010100:Oxygen