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). ¹H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe­(III) is coordinated to the 22<i>R</i>-O¹H of 22-HC, where the ¹H 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 (¹O₂) 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 ¹O₂ generation within the protein matrix. The quantum yield of ¹O₂ generation was found to be equal to 0.9 ± 0.1. On the average, six from every 10 ¹O₂ molecules succeed in escaping from the protein matrix into the solvent. A kinetic model for ¹O₂ generation within the protein matrix and for a subsequent ¹O₂ deactivation was introduced and discussed. Rate constants for radiative and nonradiative ¹O₂ deactivation within the protein were determined. The first-order radiative rate constant for ¹O₂ 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 ¹O₂ 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