tx3002753_si_001.pdb (664.48 kB)
Reaction of Human Cytochrome P450 3A4 with Peroxynitrite: Nitrotyrosine Formation on the Proximal Side Impairs Its Interaction with NADPH-Cytochrome P450 Reductase
dataset
posted on 2012-12-17, 00:00 authored by Hsia-lien Lin, Cesar Kenaan, Haoming Zhang, Paul F. HollenbergThe 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.