title: P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes.
abstract: The inactivations of P450 2B4 and the T302A mutant of 2B4 by tert-butyl acetylene (tBA) and the inactivation of 2B4 T302A by tert-butyl 1-methyl-2-propynyl ether (tBMP) have been investigated. tBA and tBMP inactivated both enzymes in a mechanism-based manner with the losses in enzymatic activity corresponding closely to losses in P450 heme. HPLC and ESI-LC-MS analysis detected two different tBA- or tBMP-modified heme products with masses of 661 and 705 Da, respectively. Interestingly, the inactivations of the P450s 2B4 by tBA and tBMP were partially reversible by dialysis, and the tBA- or tBMP-modified heme products could only be observed with ESI-LC-MS/MS when the inactivated samples were acidified prior to analysis, indicating a requirement for protons in the formation of stable heme adducts in both the wild-type and mutant 2B4 enzymes. Results of studies using artificial oxidants to support enzyme inactivation suggest that the oxenoid-iron activated oxygen species is preferentially utilized during the inactivation of the P450s 2B4 by tBA. These results argue against the use of a peroxo-iron species by P450 2B4 T302A. Molecular dynamics studies of wild-type P450 2B4 reveal that contiguous hydrogen bond networks, including structural waters, link a conserved glutamate (E301) to the distal oxygen of the peroxo-heme species via threonine 302. Interestingly, models of 2B4 T302A reveal that a compensatory, ordered hydrogen bond network forms despite the removal of T302. These results indicate that while T302 may play a role in proton delivery in the formation of the oxenoid-iron complex and in the stabilization of acetylene heme adducts in 2B4, it is not essential for proton delivery given the presence of E301 in the binding site.
authors.affiliation: Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Blobaum
authors.forename: Danni L
authors.initials: DL
authors.lastname: Harris
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D000409:Alanine / Q000235:genetics*; D000480:Alkynes / Q000737:chemistry*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004987:Ethers / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016877:Oxidants / Q000737:chemistry; D010545:Peroxides / Q000737:chemistry; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000235:genetics*
chemical_list: D000480:Alkynes; D004791:Enzyme Inhibitors; D004987:Ethers; D016877:Oxidants; D010545:Peroxides; C489139:t-butyl acetylene; C493248:tert-butyl 1-methyl-2-propynyl ether; D013912:Threonine; D006418:Heme; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D000409:Alanine