title: Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy.
abstract: We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Tosha
authors.forename: Norio
authors.initials: N
authors.lastname: Kagawa
authors.forename: Miharu
authors.initials: M
authors.lastname: Arase
authors.forename: Michael R
authors.initials: MR
authors.lastname: Waterman
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
mesh_terms: D000818:Animals; D002417:Cattle; D006861:Hydrogen Peroxide / Q000378:metabolism; D006868:Hydrolysis; D008024:Ligands; D010100:Oxygen / Q000378:metabolism*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013255:Steroid 21-Hydroxylase / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D006861:Hydrogen Peroxide; D013255:Steroid 21-Hydroxylase; D010100:Oxygen