Abstract
Cytochrome P450 4B1 (4B1) functions in both xenobiotic and endobiotic metabolism. An ester linkage between Glu-310 in 4B1 and the 5-methyl group of heme facilitates preferential hydroxylation of terminal (ω) methyl groups of hydrocarbons (HCs) and fatty acids compared with ω-1 sites bearing weaker C-H bonds. This preference is retained albeit diminished 4-fold for the E310A mutant, but the reason for this is unclear. Here, a crystal structure of the E310A-octane complex disclosed that noncovalent interactions maintain heme deformation in the absence of the ester linkage. Consistent with the lower symmetry of the heme, resonance Raman (RR) spectroscopy revealed large enhancements of RR peaks for high-spin HC complexes of 4B1 and the E310A mutant relative to P450 3A4. Whereas these enhancements were diminished in RR spectra of a low-spin 4B1-N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine complex, a crystal structure indicated that this inhibitor does not alter heme ruffling. RR spectra of Fe2+-CO HC complexes revealed larger effects of HC length in E310A than in 4B1, suggesting that reduced rigidity probably underlies increased E310A-catalyzed (ω-1)-hydroxylation. Diminished effects of the HC on the position of the Fe-CO stretching mode in 4B1 suggested that the ester linkage limits substrate access to the CO. Heme ruffling probably facilitates autocatalytic ester formation by reducing inhibitory coordination of Glu-310 with the heme iron. This also positions the 5-methyl for a reaction with the proposed glutamyl radical intermediate and potentially enhances oxo-ferryl intermediate reactivity for generation of the glutamyl radical to initiate ester bond formation and ω-hydroxylation.
Highlights
Cytochrome P450 4B1 (4B1) functions in both xenobiotic and endobiotic metabolism
To better understand the effects of the E310A mutation on the active site architecture, heme conformation, and substrate binding, the structure of the octane complex of the E310A mutant was determined by X-ray crystallography to a limiting resolution of 2.675 Å with an R/Rfree of 18.9%/23.8% (Table 1) using conditions like those used for WT 4B1 [20]
In the absence of Glu-310 and the ester linkage with the 5-methyl of the heme, the Ala-310 C␦ is positioned differently relative to the C␦ of the esterified glutamic acid, and Leu-122 shifts into a portion of the space that would otherwise be occupied by the glutamic acid ester
Summary
The 4B1 E310A mutant retains a preference for octane -hydroxylation, albeit with a 4-fold loss in /–1 regioselectivity compared with the WT enzyme while maintaining a similar overall rate of metabolite formation. These observations suggest that other aspects of the active site contribute to preferences for -hydroxylation [20]. To identify features that contribute to -hydroxylation of octane in the absence of the ester linkage, the structure of E310A mutant complexed with octane was determined and compared with the WT 4B1 structure. Together with the previously determined X-ray crystal structure of the 4B1– octane complex, the RR studies and newly determined structures of the 4B1–HET0016 and 4B1–E310A octane complexes reported here contribute to our understanding of the structural adaptations evolved to enable -regioselectivity and selective inhibition by HET0016
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