Evidence That Compound I Is the Active Species in
Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol
to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies
Cytochrome
P450scc (CYP 11A1) catalyzes the conversion of cholesterol
(Ch) to pregnenolone, the precursor to steroid hormones. This process
proceeds via three sequential monooxygenation reactions: two hydroxylations
of Ch first form 22(<i>R</i>)-hydroxycholesterol (HC) and
then 20α,22(<i>R</i>)-dihydroxycholesterol (DHC);
a lyase reaction then cleaves the C20–C22 bond to form pregnenolone.
Recent cryoreduction/annealing studies
that employed electron paramagnetic resonance (EPR)/electron nuclear
double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) <i>J. Am. Chem. Soc. 134</i>, 17149] showed that compound I (Cpd
I) is the active intermediate in the first step, hydroxylation of
Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize
the intermediates in the second and third steps of the enzymatic process,
as conducted by 77 K radiolytic one-electron cryoreduction and subsequent
annealing of the ternary oxy-cytochrome P450scc complexes with HC
and DHC. This procedure is validated by showing that the cryoreduced
ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically
competent and during annealing generate DHC and pregnenolone, respectively.
Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K
produces the superoxo-ferrous P450scc intermediate along with a minor
fraction of ferric hydroperoxo intermediates. The superoxo-ferrous
intermediate converts into a ferric-hydroperoxo species after annealing
at 145 K. During subsequent annealing at 170–180
K, the ferric-hydroperoxo intermediate converts to the
primary product complex with the large solvent kinetic isotope effect
that indicates Cpd I is being formed, and <sup>1</sup>H ENDOR measurements
of the primary product formed in D<sub>2</sub>O demonstrate that Cpd
I is the active species. They show that the primary product contains
Fe(III) coordinated to the 20-O<sup>1</sup>H of DHC with the <sup>1</sup>H derived from substrate, the signature of the Cpd I reaction.
Hydroperoxo ferric intermediates are the primary species formed during
cryoreduction of the oxy-P450scc-DHC
ternary complex, and they decay at 185 K with a strong solvent kinetic
isotope effect to form low-spin ferric
P450scc. Together, these observations indicated that Cpd I also is
the active intermediate in the C20,22 lyase final step. In combination
with our previous results, this study thus indicates that Cpd I is
the active species in each of the three sequential monooxygenation
reactions by which P450scc catalytically converts Ch to pregnenolone