期刊
BIOCHEMISTRY
卷 44, 期 10, 页码 3831-3844出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi0478953
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资金
- NCI NIH HHS [CA 16954] Funding Source: Medline
- NIDA NIH HHS [DA 017029] Funding Source: Medline
- NIDCD NIH HHS [R43-DC-6925] Funding Source: Medline
- NIGMS NIH HHS [GM 07767] Funding Source: Medline
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.
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