2 research outputs found
Compound I Is the Reactive Intermediate in the First Monooxygenation Step during Conversion of Cholesterol to Pregnenolone by Cytochrome P450scc: EPR/ENDOR/Cryoreduction/Annealing Studies
Cytochrome P450scc (CYP11A1) catalyzes conversion of
cholesterol
(CH) to pregnenolone, the precursor to all steroid hormones. This
process proceeds via three sequential monooxygenation reactions: two
stereospecific hydroxylations with formation first of 22<i>R</i>-hydroxycholesterol (22-HC) and then 20α,22<i>R</i>-dihydroxycholesterol (20,22-DHC), followed by C20–C22 bond
cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize
the intermediates in the first hydroxylation step by 77 K radiolytic
one-electron cryoreduction and subsequent annealing of the ternary
oxy-cytochrome P450scc-cholesterol complex. This approach is fully
validated by the demonstration that the cryoreduced ternary complex
of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol
to form 22-HC with no detectable formation of 20-HC, just as occurs
under physiological conditions. Cryoreduction of the ternary complex
trapped at 77 K produces predominantly the hydroperoxy-ferriheme P450scc
intermediate, along with a minor fraction of peroxo-ferriheme intermediate
that converts into a new hydroperoxo-ferriheme species at 145 K. This
behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol
complex exhibits an efficient proton delivery network, with an ordered
water molecule H-bonded to the distal oxygen of the dioxygen ligand.
During annealing of the hydroperoxy-ferric P450scc intermediates at
185 K, they convert to the primary product complex in which CH has
been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate
decays with a large solvent kinetic isotope effect, as expected when
proton delivery to the terminal O leads to formation of Compound I
(Cpd I). <sup>1</sup>H ENDOR measurements of the primary product
formed in deuterated solvent show that the heme FeÂ(III) is coordinated
to the 22<i>R</i>-O<sup>1</sup>H of 22-HC, where the <sup>1</sup>H is derived from substrate and exchanges to D after annealing
at higher temperatures. These observations establish that Cpd I
is the agent that hydroxylates CH, rather than the hydroperoxy-ferric
heme
Photosensitized Singlet Oxygen Luminescence from the Protein Matrix of Zn-Substituted Myoglobin
A nanosecond laser near-infrared
spectrometer was used to study
singlet oxygen (<sup>1</sup>O<sub>2</sub>) emission in a protein matrix.
Myoglobin in which the intact heme is substituted by Zn-protoporphyrin
IX (ZnPP) was employed. Every collision of ground state molecular
oxygen with ZnPP in the excited triplet state results in <sup>1</sup>O<sub>2</sub> generation within the protein matrix. The quantum yield
of <sup>1</sup>O<sub>2</sub> generation was found to be equal to 0.9
± 0.1. On the average, six from every 10 <sup>1</sup>O<sub>2</sub> molecules succeed in escaping from the protein matrix into the solvent.
A kinetic model for <sup>1</sup>O<sub>2</sub> generation within the
protein matrix and for a subsequent <sup>1</sup>O<sub>2</sub> deactivation
was introduced and discussed. Rate constants for radiative and nonradiative <sup>1</sup>O<sub>2</sub> deactivation within the protein were determined.
The first-order radiative rate constant for <sup>1</sup>O<sub>2</sub> deactivation within the protein was found to be 8.1 ± 1.3 times
larger than the one in aqueous solutions, indicating the strong influence
of the protein matrix on the radiative <sup>1</sup>O<sub>2</sub> deactivation.
Collisions of singlet oxygen with each protein amino acid and ZnPP
were assumed to contribute independently to the observed radiative
as well as nonradiative rate constants