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Characterization of mutants of three highly conserved amino acids in microsomal cytochrome P450 2B4
Author(s) -
Waskell Lucy,
Zhang Haoming,
Im SangChoul,
Davydov Roman,
Hoffman Brian
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a459-a
Subject(s) - heme , mutant , histidine , chemistry , amino acid , cysteine , cytochrome , biochemistry , cytochrome p450 , hemeprotein , mutant protein , cytochrome c , lysine , stereochemistry , enzyme , gene , mitochondrion
Three highly conserved amino acids in cytochrome P450 2B4 (CYP2B4) were mutated: E301Q, T302A, and F429H. The mutant proteins were expressed in E. coli and extensively characterized. T302 and E301 are on the distal side of the heme and likely play a key role in proton delivery as well as its regulation whereas F429 is on the proximal side of the heme in the beta‐bulge near the axial cysteine. Catalytic activity was impaired by 83–93% for each of the 3 mutant proteins. The T302A mutant protein exhibited behavior consistent with the hydroxyl group of T302 hydrogen bonding to the ligands that successively bind the heme during catalytic turnover (H 2 O, O 2 , − OOH). Mutation of F429 to histidine markedly altered the electronic properties and reactivity of the heme and may lead to a conformational change on the proximal surface of CYP2B4. The F429H protein demonstrated an increased redox potential and paradoxically a slower rate of reduction of the ferric protein. An EPR spectrum was atypical and the mutant protein auto oxidized slowly compared to the wild type. This research was supported by NIH Grant (GM035533) and a VA Merit Grant.