Autocatalytic mechanism and functional consequences of covalent heme attachment in CYP4B1

ResearchWorks/Manakin Repository

Search ResearchWorks

Advanced Search


My Account


Related Information

Autocatalytic mechanism and functional consequences of covalent heme attachment in CYP4B1

Show full item record

Title: Autocatalytic mechanism and functional consequences of covalent heme attachment in CYP4B1
Author: Baer, Brian R. (Brian Ray), 1977-
Abstract: The studies presented in this dissertation establish the precise structure and sites of attachment of the covalent linkage that binds heme to CYP4B1, reveal a viable autocatalytic mechanism for its formation, and evaluate functional consequences on enzymatic activity and physical properties of this novel, structurally modified P450. The site of protein attachment in CYP4B1 was identified as Glu310 in the I-helix, via mass analysis of heme-containing tryptic peptides, sequence alignments, and site-directed mutagenesis. Two-dimensional NMR and chromatographic analysis of base-hydrolyzed hemes revealed that native CYP4B1 heme was modified solely at the C-5 methyl, but recombinant CYP4B1 heme was also modified at the C-8 methyl. These results are indicative of two heme orientations in the active site of recombinant P450s, yet further experiments demonstrated that this microheterogeneity of heme orientation may not affect in vitro substrate kinetics. The CYP4B1 E310D mutant produced free hydroxymethylheme, suggesting that the mechanism for the formation of the ester bond involved a heme methyl carbocation intermediate. To investigate the mechanism further, the carboxylates of glutamic acid residues were labeled with 18O in the wild type enzyme and the fate of the heavy isotope was traced into base-hydrolyzed monohydroxyhemes by LC/ESI-MS. These data support a mechanism that parallels the stepwise reaction proposed for the covalent attachment in the mammalian peroxidases, namely initial hydrogen abstraction from the carboxylate of Glu310 to form a carboxyl radical, hydrogen abstraction from the heme methyl, intramolecular electron transfer to generate a heme methyl carbocation, and finally, quenching of the carbocation with the same carboxylate of Glu310. CYP4B1 wild-type and the E310 mutants, devoid of the covalent heme link, were compared in functional assays with lauric acid, various hydrocarbons, 4-ipomeanol, pyrogallol, and 1,2,4,5-tetramethoxybenzene, but no obvious distinction emerged. CYP4B1 was comparable to the E310A mutant in terms of heme retention and thermostability, but polar substitutions were not well tolerated in the thermostability assay. Therefore, during the evolution of the CYP4B1 gene, a random A310E mutation may not have unduly perturbed the selective pressures imposed on the P450 because the resulting polar glutamic acid could be masked by incorporation into the ester bond.
Description: Thesis (Ph. D.)--University of Washington, 2005.

Files in this item

Files Size Format View
3198768.pdf 9.908Mb PDF View/Open

This item appears in the following Collection(s)

Show full item record