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Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

ABSTRACT: The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO(3+)). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using (18)O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen ((18)O2), and coupled assays were used to trap the labile (18)O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the (18)O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an (18)O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.

SUBMITTER: Yoshimoto FK

PROVIDER: S-EPMC5289895 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

Yoshimoto Francis K FK Jung I-Ji IJ Goyal Sandeep S Gonzalez Eric E Guengerich F Peter FP

Journal of the American Chemical Society 20160912 37

The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO(3+)). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol ...[more]

PMID: 27571509

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Project description:The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.

Dataset Information

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

Publications

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

Similar Datasets

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