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 17␣-hydroxyprogesterone. The uncoupled H 2 O 2 formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17␣-hydroxyprogesterone (15% of NADPH consumed), indicating that 17␣-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode ( O-O ) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone-or 17␣-hydroxyprogesterone-bound enzyme gave single (at 1137 cm ؊1 ) or split O-O bands (at 1124 and 1138 cm ؊1 ), respectively, demonstrating the presence of two forms for the latter. In contrast to 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 substratebound 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 17␣-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower 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 17␣-hydroxyprogesterone than in progesterone.Cytochrome P450 steroid 21-hydroxylase (P450c21) 4 is predominantly expressed in adrenal cortex under the control of adrenocorticotropic hormone via the cAMP-dependent signaling pathway (1, 2). P450c21 catalyzes hydroxylation at C-21 of progesterone (Prog) and 17␣-hydroxyprogesterone (17-OHprog) to produce 11-deoxycorticosterone and 11-deoxycortisol, respectively, as shown in Fig. 1. These C-21 hydroxylation steps are essential for the biosynthesis of aldosterone and cortisol. Deficiency in P450c21 found in ϳ1:10,000 newborn babies results in impaired steroid synthesis in which adrenal androgens are overproduced in addition to the lack of aldosterone and cortisol, as shown in Fig. 1 (3). The P450c21 deficiency accounts for more than 90% of a common genetic disease, congenital adrenal hyperplasia, with symptoms of salt wasting, simple virilizing, and nonclassical phenotypes (3). The enzyme deficiency can be attributed to mutations in the CYP21B gene encoding P450c21 (4, 5). For better understanding the mechanism of the deficiency of P450c21 by the mutation, it is highly desirable to elucidate the structure-function relationship in P450c21.The structural and functional studies have been carried out with use of bovine P450c21 purified from adrenal cortex (6). P450c21 shows higher hydroxylation activity with 17-OH-prog than that of...

Resonance Raman Spectroscopy Reveals That Substrate Structure Selectively Impacts the Heme-Bound Diatomic Ligands of CYP17

“…Finally, it is noted that the 347 cm −1 value observed for the ν(Fe-S) mode is at the low end of values obtained for other cytochromes P450, which range between ~353 down to 347 cm −1 . 29,32,49-52 As will be seen, this lower ν(Fe-S) has an impact on the ν(Fe-C) and ν(C-O) modes of the trans-axial Fe-C-O fragment (vide infra).…”

Oxygen activation by cytochrome P450 monooxygenase

“…The involvement of the hydroxyl group of the substrate in proton delivery was reported for P450c21, P450 107A, and P450 158A2 (Nagano et al, 2005; Zhao et al, 2005; Tosha et al, 2008). P450c21 catalyzes the hydroxylation of progesterone and 17α-hydroxyprogesterone at C-21 position and the reaction is better coupled with 17α-hydroxyprogesterone by 11%.…”

“…P450c21 catalyzes the hydroxylation of progesterone and 17α-hydroxyprogesterone at C-21 position and the reaction is better coupled with 17α-hydroxyprogesterone by 11%. The studies of the vibrational mode of the oxyferrous P450c21 by Raman spectroscopy demonstrated that ν O-O is sensitive to the substrate; progesterone gave a single ν O-O band at 1137 cm −1 while 17α-hydroxyprogesterone split ν O-O into two bands at 1124 and 1138 cm −1 (Tosha et al, 2008). It was postulated that 17α-hydroxyprogesterone is hydrogen bonded to the iron-linked oxygen and participates in proton delivery.…”

Substrate binding to cytochromes P450

“…Substrate binding to P450s 102A1 [123], 19A1 (aromatase) [124], 2B4 [125], and 2D6 [126] has been studied with resonance Raman spectroscopy. Recently the differences between oxidation efficiency of two endogenous substrates of P450 21A1 (steroid 21-hydroxylase), progesterone and 17α-hydroxyprogesterone, have been explained with the aid of resonance Raman spectroscopy studies [127]. P450-substrate interactions have also been studied on self-assembled monolayer (SAM)-coated metal surfaces (silver and gold) by resonance Raman scattering spectroscopy [126, 128] where the sensitivity is increased via so called surface enhancement [129].…”