Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition

Article abstract of DOI:10.1074/jbc.M117.792200

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl) phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H₂O₂ attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H₂O₂ concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.

Full text of DOI:10.1074/jbc.M117.792200

JBC Papers in Press. Published on May 22, 2017 as Manuscript M117.792200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.792200
P450 4A11 Sulfenic Acid and Inhibition
Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic
inhibition*S
Matthew E. Albertolle1, Donghak Kim1,2, Leslie D. Nagy1, Chul-Ho Yun3, Ambra Pozzi4,5, Üzen
Savas6, Eric F. Johnson6, and F. Peter Guengerich1
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-
0146, 2Department of Biological Sciences, Konkuk University, Seoul 05025, Republic of Korea, 3School
of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of
Korea, 4Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-
6602, 5Veterans Affairs Medical Center, Nashville, Tennessee, 37232, and 6Department of Molecular
Medicine, The Scripps Research Institute, La Jolla, California 92037
Running title: P450 4A11 Sulfenic Acid and Inhibition
Address correspondence to: Prof. F. Peter Guengerich, Department of Biochemistry, Vanderbilt
University School of Medicine, 638B Robinson Research Building, 2200 Pierce Avenue, Nashville,
Tennessee 37232-0146, Telephone: (615) 322-2261; FAX: (615) 343-0704; E-mail:
f.guengerich@vanderbilt.edu
Keywords: Cytochrome P450, mass spectrometry, proteomics, thiol, oxidative damage
ABSTRACT
Cytochrome P450 (P450, CYP) 4A11 is a
agents, including dithiothreitol and dithionite. Of
note, we observed heme ligand cysteine
sulfenylation of P450 4A11 ex vivo in kidneys and
livers derived from CYP4A11 transgenic mice. We
also detected sulfenylation of murine P450s 4a12
and 4b1 heme peptides in kidneys. To our
knowledge, reversible oxidation of the heme
thiolate has not previously been observed in
P450s, to our knowledge, and may have relevance
for 20-HETE-mediated functions.
human fatty acid ω-hydroxylase that catalyzes the
oxidation of arachidonic acid to the eicosanoid 20-
hydroxyeicosatetraenoic acid (20-HETE), which
plays important roles in regulating blood pressure
regulation. Variants of P450 4A11 have been
associated with high blood pressure and resistance
to anti-hypertensive drugs, and 20-HETE has both
pro- and antihypertensive properties relating to
increased vasoconstriction and natriuresis,
respectively. These physiological activities are
likely influenced by the redox environment, but
the mechanisms are unclear. Here, we found that
reducing agents (e.g., dithiothreitol and
tris(carboxyethyl)phosphine) strongly enhanced
the catalytic activity of P450 4A11, but not of 10
other human P450s tested. Conversely, added
H2O2 attenuated P450 4A11 catalytic activity.
Catalytic roles of five of the potentially eight
implicated Cys residues of P450 4A11 were
eliminated by site-directed mutagenesis. Using an
isotope-coded dimedone/iododimedone labeling
strategy and mass spectrometry of peptides, we
demonstrated that the heme-thiolate cysteine (Cys-
457) is selectively sulfenylated in a H2O2
Human cytochrome P450 (P450, CYP)
enzymes catalyze oxidations of numerous
endogenous substrates, including eicosanoids,
steroids, and vitamins (1). At least 13 human P450
enzymes (2C8, 2C9, 2J2, 2U1, 4A11, 4F22, 4F12,
4V2, 4F2, 4F3, 4F8, 5A1, and 8A1) utilize fatty
acids and/or eicosanoids as substrates (2). P450
4A11 is primarily an ω-hydroxylase, with its main
endogenous role apparently being the conversion
of arachidonic acid to 20-hydroxyeicosatetraenoic
acid (20-HETE) (3). P450 4A11 can also catalyze
oxidations of other fatty acid substrates, including
lauric acid (4):
CH3(CH2)nCO2H —> HOCH2(CH2)nCO2H
P450 4A11 is expressed mainly in the
liver, kidney, and vasculature (5,6), and Subfamily
concentration-dependent
manner.
This
sulfenylation could be reversed by reducing
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

P450 4A11 Sulfenic Acid and Inhibition
4A P450 enzymes have been associated with
vascular dysfunction and hypertension in rodent
models and humans (7). The P450 4A11 F434S
variant has been associated with increased blood
pressure in several populations studied (8-13). 20-
HETE plays opposing roles, having both pro- and
enzymes might have relevance to the local
production of 20-HETE.
RESULTS
Stimulation
of
P450
4A11
ω-
anti-hypertensive
actions
by
promoting
Hydroxylation Activity by Reducing Agents—In
previous work the rates of w-hydroxylation of
lauric acid varied considerably, from 9-21 min-1,
under varying conditions (4). When P450 4A11
was pretreated with either dithiothreitol (DTT) or
tris(carboethoxy)phosphine (TCEP) reagents in
our preliminary experiments (Fig. 1), ω-
hydroxylation activity was routinely increased
two- to four-fold. Similar results were found with
reduced glutathione (results not presented). The
ratio of the two major products, 11- and 12-
hydroxy lauric acid, remained constant with this
activation (results not presented). These results
indicated that P450 4A11 has a thiol-dependent
redox sensitivity, which was investigated further.
Redox-dependent activation was also reflected in
the increase in kcat values for both the wild-type
and F434S (rs1126742) (8) variants of P450 4A11
(2.1-fold) (Figs. 2A, 2B). The Km values with and
without DTT remained constant between the wild-
type enzyme (63 ± 10 µM and 62 ± 15 µM,
respectively) and the F434S variant (67 ± 7 µM
and 58 ± 10 µM, respectively) (Figs. 2A, 2B).
Collectively, these results indicate that both wild-
type and F434S variant P450 4A11 are activated in
vasoconstriction or natriuresis, respectively (14).
20-HETE has been reported to induce transcription
of NADPH oxidase, activating this enzyme
complex through protein kinase C (15-18), and has
also been reported to increase mitochondrial
reactive oxygen species (ROS) production (19).
Studies with P450 4A11 transgenic mice have
identified P450 4A11 regulation via fasting and
growth hormone (20) and that 20-HETE and P450
4A11 activity may uncouple the renal renin
angiotensin system (21).
ROS, specifically H2O2, have recently
become regarded as important secondary signaling
molecules (22). H2O2 modifies epidermal growth
factor receptor signaling (23) and regulates
glycolysis through glyceraldehyde-3-phosphate
dehydrogenase (24). Common targets of H2O2 are
protein thiols, oxidizing them to more reactive
species, mainly sulfenic acids (-SOH). This
transient posttranslational modification aids in the
formation of inter- or intra-molecular disulfide
bonds, alters cellular signaling, and regulates
enzymatic activity (25,26).
In this study, we noted the stimulation of
P450 4A11 activity by thiols and other reducing
agents and further examined the ability of P450
4A11 to respond to perturbations in the redox
environment in vitro. Ten other recombinant
human P450s were tested for sensitivity to
reductants and no large changes in activity were
noted. Five of the eight possible cysteine (to
serine) mutants of P450 4A11 were expressed and
were found to be catalytically active, reducing the
possibilities for the sensitive thiol. We observed
that P450 4A11 is sensitive to oxidation and,
interestingly, sulfenic acid formation was able to
disrupt binding of Cys-457 to the heme iron and
inactivate the enzyme, in a very reversible process.
We also detected sulfenylation of P450 4A11 and
murine Subfamily 4a/b P450 enzymes in kidneys
and livers derived from CYP4A11 transgenic mice,
suggesting that changes in the redox state of these
a
reducing environment, which may have
biological relevance in vivo.
Ten other human P450 enzymes were
tested for thiol-dependent activation with their
relevant substrates (Table 1). The highest
stimulation observed among these was P450 2C9,
with only a 33% increase. The redox sensitivity of
P450 4A11 was unique among human P450
enzymes examined here.
Oxidative Inhibition of P450 4A11—P450
4A11 hydroxylation activity was inhibited by
H2O2. This inhibition occurred in a time-dependent
manner with an 80% loss in activity occurring
within 15 minutes (Fig. 3), a rate much higher than
reported for uncatalyzed sulfenylation of thiols
(27). Fifty percent activity loss occurred at a
concentration of 140 µM H2O2 (Fig. 4).
2

P450 4A11 Sulfenic Acid and Inhibition
Site-directed Mutagenesis—P450 4A11
encodes eight cysteinyl residues in the translated
protein, and five of these cysteines were mutated
to serines. Cys-457 corresponds to the proximal
heme ligand and could not be changed (28), and
neither C347S nor C375S produced protein with
the typical P450 difference spectrum (29). Steady-
state kinetic assays were done with the remaining
five cysteine mutants, both with and without DTT
pretreatment (Fig. 5). The response to DTT was
qualitatively the same as the wild-type P450 4A11
(Fig. 2A), except for C200S (Fig. 5C) and possibly
C513S (Figs. 5A, 5E), but these analyses did not
implicate any of these five cysteines as being
involved in the modulation of hydroxylation
activity observed in the wild-type enzyme. The
major conclusion was that none of these five
cysteines are critical to the normal activity of P450
450 4A11, leaving only the non-mutated Cys-347,
Cys-375, and Cys-457 as candidates for the
oxidation-reduction phenomenon.
resuspended, and treated with d5-iodoacetanilide to
alkylate free thiols (35). The protein was then
precipitated and resuspended in reducing buffer to
reduce disulfide bonds (and any unreacted sulfenic
acids) and alkylated with d0-iodoacetanilide.
Tryptic peptides were analyzed via LC-MS/MS
and the area ratios of alkylated deuterated/non-
deuterated thiol containing peptides were
analyzed. This experiment showed no differences
in ratios between a reduced P450 4A11 protein
control and up to 1 mM H2O2 (Supplemental Data
Fig. S2). Seven of the eight cysteines in P450
4A11 maintained at least 60% reduction (i.e.,
>60% d0 labeling) with only Cys-513, the
carboxy-terminal cysteine, being oxidized further
with increasing H2O2.
Spectral Analysis of Heme-thiolate
Oxidation—Spectral properties of P450 4A11
were used to further address the proteomic results,
i.e. to address the hypothesis that the sulfenic acid
moiety is disrupting the iron coordination of the
heme, TCEP-treated P450 4A11 was treated with
H2O2, reconstituted with NADPH-P450 reductase,
deaerated, and placed under an anaerobic
atmosphere of CO. Following the addition of
NADPH, spectra were recorded for TCEP-reduced
(Fig. 9A) and H2O2-oxidized (Fig. 9B) P450 4A11.
The H2O2-oxidized protein showed increased
absorbance at 420 nm for the CO-bound form.
Sodium dithionite was added to these samples,
which increased the absorbance of the oxidized
enzyme at 450 nm and decreased the absorbance at
420 nm. NADPH-P450 reductase is able to reduce
ferric heme to the ferrous state (allowing CO
binding) but cannot reduce the sulfenic acid,
leading to an inactive protein due to the loss of the
thiolate axial coordination. However, dithionite is
able to reduce a sulfenic acid (36), allowing for the
reduced thiolate to re-ligand with the heme-iron
(Fig. 10). The change in A450 upon the addition of
dithionite in Fig. 9B corresponds to an increase of
~ 0.3 µM P450 (~ 30% total, uncorrected for loss
of P450 in the degassing procedure), somewhat
less than predicted from the LC-MS labeling study
(Fig. 8).
Identification of
a Sulfenic Acid—A
portion of oxidized P450 4A11 treated with each
of several different concentrations of H2O2 was
treated with d6-dimedone (co-incubation) and
subsequently
counter-alkylated
with
(d0-)
iododimedone (30) (Fig. 6). The alkylated protein
was digested with trypsin and analyzed by LC-
MS/MS (Fig. 7); integrated areas of extracted ion
chromatograms for the heavy (sulfenic acid-
modified) and light (free thiol) were compared
using the program Skyline (31). Sulfenylation of
(the heme-thiolate ligand) Cys-457 coincided with
the attenuation of enzymatic activity observed
with increasing concentrations of H2O2 (Fig. 8,
Supplemental Fig. S1, S2). Oxidation of the heme-
thiolate ligand would lead to an inability of the
heme iron to effectively activate oxygen and carry
out catalysis.
This sulfenylation, however, did not
exclude the possibility for thiol oxidation to occur
in the form of intra- or intermolecular disulfide
bonds for other P450 4A11 thiols. An isotope-
coded affinity tagging (ICAT) approach was
developed using d5/d0-iodoacetanilide (32,33) (Fig.
6). Briefly, samples were treated with varying
concentrations of H2O2 and thiols were then
trapped using a trichloroacetic acid precipitation
(34). Protein was then pelleted by centrifugation,
Sulfenylation of Family 4 P450 Enzymes
in a CYP4A11 Transgenic Mouse Model—To
determine if this oxidative modification of P450
4A11 occurs in vivo, a CYP4A11 transgenic mouse
3

P450 4A11 Sulfenic Acid and Inhibition
model was used (20). Liver and kidney tissues
from four male mice were harvested and
subcellular fractionation was done immediately.
Microsomes were obtained within three hours of
tissue harvesting via differential centrifugation
and, as with the recombinant protein, incubated
with either buffer, d6-dimedone, or d6-dimedone
supplemented with 500 µM H2O2 (Fig. 6). These
labeled samples were then treated with catalase,
It is well-established that breaking this bond will
also convert P450 to an inactive form, cytochrome
P420 (38,39). The accessibility of the heme
thiolate ligand has been established in studies with
mercurials, as well as the conversion to the
inactive cytochrome P420 (40,41).
The concentration of H2O2 required to
inhibit P450 4A11 (Fig. 4) is within
a
physiological range (42) and was shown to be
reversible via reduction by thiols (Fig. 1). P450
enzymes are known to produce H2O2 in
conjunction with NADPH-P450 reductase and
NADPH, with or without substrate present (43).
H2O2 production was measured (43) for a typical
reaction containing 0.2 µM P450 4A11, 0.4 µM
NADPH-P450 reductase, the NADPH-generating
system, and 100 µM lauric acid and determined to
be 5 µM min-1, i.e. 25 nmol H2O2 produced min-1
(nmol P450 4A11)-1. Typical incubations with
lauric acid or arachidonic acid are conducted for
two to three times this long, and the concentration
of H2O2 produced should be negligible in
proportion to the amount needed to inhibit the
enzyme (Fig. 4).
reduced,
counter-alkylated
with
(d0-)
iododimedone, and digested with chymotrypsin.
Chymotrypsin was used to produce peptides
containing the heme-ligand cysteine unique to
both P450s 4A11 and 4a12 in kidneys of male
mice (Supplemental Fig. S3) (37). The microsomal
chymotryptic peptides were analyzed via LC-
MS/MS and the resulting peak area ratios were
quantitated. The heme-thiolate Cys-457 of P450
4A11 showed
~
75% dimedone labeling
(sulfenylation) in both the dimedone and H2O2-
supplemented samples in the kidney microsomes
of three mice and ~40% labeling in the liver
microsomes (Figs. 11A, 11B, Supplemental Data
Fig. S4). One mouse was considerably younger
(age of 6 weeks at time of harvest, compared to
the other three (two of which which were 3
months old and one was 7 months old) and the
expression of P450 4A11 was greatly diminished
compared to the older mice, as judged by peptide
recovery. Interestingly, murine P450s 4a12 (Fig.
11C, 11D, Supplemental Data Fig. S5) and 4b1
(Fig. 11E, Supplemental Data Fig. S6) also
showed significant sulfenylation of the respective
heme-thiol ligands. The younger mouse had
consistently lower levels of sulfenylation of the
heme binding peptide for all of the Family 4 P450s
in the kidneys and lower levels of P450 4a12 in
the liver, but the sulfenylation of P450 4A11 in the
liver was not different form the older mice.
Thiol activation is common in in vitro
(25). The activation seen in the case of P450 4A11
was considerably higher than with any other P450s
tested (Table 1), indicating that this redox
sensitivity is rather unique among the human
P450s tested. Although numerous P450
purification protocols include dithiothreitol or b-
mercaptoethanol (44,45), it is not clear that this is
necessary in most cases.
Oxidation of P450 4A11 with H2O2 led to
an attenuation of enzymatic activity (Figs. 3, 4).
This loss of activity was correlated with an
increased observation of dimedone alkylation
(sulfenylation) at Cys-457, the heme-thiolate
ligand (Figs. 7, 8). Sulfenic acid modifications
have been directly implicated in the regulation of
DISCUSSION
numerous
enzymatic
processes
including
We have shown that H2O2 reacts with the
heme-thiolate cysteine of P450 4A11 to form a
sulfenic acid and disrupt the iron-sulfur
coordination. Five of the eight Cys residues in the
protein were shown not to be essential for activity
(Fig. 5), and the heme proximal ligand Cys-457
was identified as the major site of oxidation by
modification and proteomic analysis (Figs. 7, 8).
glyceraldehyde-3-phosphate
dehydrogenase,
peroxiredoxin, epidermal growth factor receptor,
and others (26). However, sulfenic acid formation
involving disruption of heme-iron coordination is
unprecedented to our knowledge. Choudhury et al.
(46) mutated the cytochrome c peroxidase distal
histidine ligand to a cysteine and observed a
cysteic acid (cysteine sulfonic acid) in a crystal
4

P450 4A11 Sulfenic Acid and Inhibition
structure of the cysteine mutant, with a sulfonate
oxygen coordinated to the heme. They observed
activity but it was markedly lower than that of the
unmodified enzyme. These results were
interpreted as the heme iron itself oxidizing the
cysteine, as a substrate. This is an appropriate
conclusion, in that peroxidases generally have
non-Cys heme ligands (47,48). Nitrile hydratase
enzymes are known to coordinate iron or cobalt
with both sulfenylated and sulfinylated thiols;
additionally, the sulfenic acid is essential for the
formation of the carbamate product (49). Our
results do not discern whether the sulfenylated
thiol is still (weakly) coordinated to the heme or if
the coordination completely disrupted in P450
4A11. Dimedone, however, can react with
sulfenamides as well as sulfenic acids (50) and
such reactions would provide similar results in
of these ancillary cysteines could produce slight
conformational changes, but these changes would
not produce the spectral change at 450 nm that
sulfenylation of Cys-457 does.
Detection of sulfenylation in murine
kidneys and livers provides a physiological
context for this modification. The high percentage
of dimedone alkylation seen in the P450 4A11,
4a12, and 4b1 heme peptides was surprising in
that this would imply that a large percentage of the
Family 4 P450 enzymes is constitutively inactive
in the kidney. One possible explanation could be
that, since 20-HETE is
a
very potent
vasoconstrictor with submicromolar activity
(52,53), these enzymes are very tightly regulated
and sulfenylated enzymes could act as a reserve
mechanism. Conversely, if an increase in vascular
tone is required quickly, Family 4 P450 enzymes
could be reduced quickly and be available for
production of 20-HETE. P450 4A11 sulfenylation
was observed to a lesser degree in liver. This may
be due to the larger reducing potential in
glutathione stores the liver has compared to the
kidney (54).
these experiments as sulfenic acid. If
a
sulfenamide was indeed the cause of this redox
modulation, generally thiol oxidation to sulfenic
acid would occur first followed by subsequent
condensation with a backbone nitrogen to form the
sulfenamide (50).
ICAT analysis with iodoacetanilide did
not provide evidence for a disulfide bond. a-
Haloketones react with sulfenic acids to form
sulfoxides (51), so these peptides would not be
quantified in a direct analysis between d0/d5-
alkylated peptides because the m/z would increase
by 16, the mass of oxygen. Inaccuracy could also
One unique feature of Subfamily 4A P450
enzymes is the covalent binding of the heme
prosthetic group to the enzyme through
a
conserved glutamic acid residue (55,56). This
unique feature may allow for easier re-
coordination of the reduced thiol with an
immobilized heme iron. In recombinant P450
4A11 it is reported that approximately 25% of the
enzyme has covalently bound heme (56), but the
fraction has not been determined in human
samples. Rodent subfamily 4A P450 enzymes
have higher percentages of heme covalently bound
to the enzyme (56). LeBrun et al. (56) suggest that
this covalent binding may contribute to differences
in substrate binding rates and enzymatic
regioselectivity.
–
be due to further di- and tri-oxidation (-SO2 and -
–
SO3 , respectively) of cysteines.
Cys-513
appeared to be the only thiol oxidized to a large
extent in our analysis, although the exact nature is
unknown. Additionally, Cys-347 and Cys-375
appear to have proximity to each other in the
homology model (Fig. 12). The inability to
express the mutated forms of these two cysteines
indicates that they are probably important for
structural and/or conformational integrity, but our
ICAT analysis does not indicate that either thiol is
oxidized.
A recent crystal structure of recombinant
rabbit P450 4B1 shows complete adduction of the
heme through a bond between a heme methyl
group and Glu-310 (analogous to Glu-321 in P450
4A11) (57). In examining the alignment of mouse
P450 4b1 and rabbit P450 4B1 (uniport.org), these
two enzymes share 86% identity and 95%
similarity. A modeled structure shows a relatively
open conformation on the proximal side of the
Taken together the mutant analysis,
dimedone labeling, and ICAT labeling methods
show that cysteines other than Cys-457 are
oxidized in P450 4A11 but do not appear to
directly inactivate the protein or are correlated
with the loss of activity. The oxidation or mutation
5

P450 4A11 Sulfenic Acid and Inhibition
protein (Fig. 12). Cysteines that were mutated in
this study are located in the peripheries of the
protein, away from the proposed active site, and
likely do not influence enzyme function.
EXPERIMENTAL PROCEDURES
Chemicals—[1-14C]-Lauric
obtained from American Research Chemicals (St,
Louis, MO). Lauric acid, TCEP, H2O2 (30% w/v),
paclitaxel, tolbutamide, bufuralol, nifedipine,
acid
was
Disruption of the iron-sulfur coordination
would alter the absorbance properties of P450
4A11 (Fig. 10). Under an anaerobic CO
atmosphere, NADPH-P450 reductase can reduce
ferric heme to its ferrous state, allowing CO to
bind. This CO-bound state has absorbance with
λmax at 450 nm if the heme-thiolate ligand is intact.
When this coordination is disrupted (via
sulfenylation in this case), the CO-binding
spectrum was shifted to a λmax at 420 nm (Figs. 9
and 10). Interestingly, the addition of dithionite
after reduction with NADPH-P450 oxidoreductase
reduces the sulfenic acid (36) and allows for re-
coordination of the thiolate-heme complex. The
observation of a sulfenylated heme-thiolate ligand
has probably been overlooked previously because
of the common laboratory practice of determining
P450 concentration using dithionite as the
reducing agent for obtaining P450 difference
spectra.
testosterone,
progesterone,
coumarin,
and
ethoxyresorufin were obtained from Sigma
Aldrich.
Chemical Synthesis
Iododimedone—Iododimedone
was
prepared from dimedone (4 mmol scale) by
iodination with N-iodosuccinimide (59). Yield
48%, mp 145-146 °C; high resolution mass
spectrometry (HRMS) for C8H12O2I m/z 266.9882
(MH+), found 266.9881 (Δ 0.4 ppm); H-NMR
1
(400 MHz, CDCl3) δ 1.04 (s, 6H, (CH3)2), 2.50 (d
(splitting due to I), 4H, CH2), 6.35 (s, 1H, CHI)
(literature mp 155 °C (60)).
d6-Dimedone—A modification of the
basic procedure of Yeo and Carroll (30) was used.
Diethyl malonate and mesityl-d10-oxide were
condensed in an ethanolic solution of NaOC2H5
under reflux, followed by decarboxylation with
NaOH under reflux. Following neutralization, the
filtrate was collected and extracted into ethyl
acetate. The product was concentrated by partial
removal of solvent in vacuo and trituration with
hexanes to yield crystalline d6-dimedone in 85%
yield. mp 147-148 °C; HRMS for C8H7D6O2 m/z
In summary, P450 4A11 seems to show a
unique activation under reducing conditions
compared to other P450 enzymes tested.
Conversely, H2O2 inhibits the enzymatic activity
of the enzyme. Sulfenic acid formation of the
heme thiolate ligand was detected by labeling and
LC-MS analysis (Figs. 7, 8) and was verified
spectrally (Fig. 9). The physiological relevance of
the modification was demonstrated with the
identification of sulfenylated P450s in freshly
isolated murine kidneys and livers. This
modification is a potentially important observation
that may have implications in other P450s, as
shown by our results with the mouse P450 4a and
4b enzymes. Sulfenylation could provide for
biological regulation of P450 4A11 and its
production of the potent vasoconstrictor 20-HETE.
As mentioned previously, 20-HETE has been
shown to induce ROS formation as well as play an
important role in blood pressure control, which is
known to be mediated by ROS production (58).
The extent of sulfenylation of 4A/a P450 enzymes
in tissues may provide a mechanism to reduce the
production of 20-HETE.
147.1292 (MH+), obs 147.1284 (Δ 5.4 ppm); H-
NMR (400 MHz, CDCl3) δ 2.52 (s, 5H, -CH2-),
3.33 (s, 2H, CO-CH2-CO) (61) (lit mp 145-147 °C
(61)).
1
d0-Iodoacetanilide—Iodoacetanilide was
prepared from iodoacetic acid (2.53 mmol) and
aniline (2.53 mmol) by amide coupling with
dicyclohexylcarbodiimide (2.53 mmol) (62). Yield
1
70%, H-NMR (600 MHz, acetone-d6) δ 9.51 (bs,
1H, NH) 7.64 (d, 2H, o-Ar), 7.31 (t, 2H, m-Ar),
7.02 (t, 1H, p-Ar), 3.90 (s, 2H, CH2I), 13C-NMR
(150 MHz, acetone-d6) 167.0, 140.1, 129.7, 124.6,
120.0, 0.72.
d5-Iodoacetanilide— d5-Iodoacetanilide
was prepared from iodoacetic acid (2.53 mmol)
and d5-aniline (2.53 mmol) by amide coupling
with dicyclohexylcarbodiimide (2.53 mmol) (62).
1
Yield 89%, H-NMR (600 MHz, acetone-d6) δ
6

P450 4A11 Sulfenic Acid and Inhibition
9.53 (bs, 1H, NH), 3.90 (s, 2H, CH2I), 13C-NMR
(150 MHz, acetone-d6) 167.0, 139.8, 129.1, 124.1,
119.6, 0.75.
(DLPC, Sigma Aldrich), 100 mM potassium
phosphate buffer (pH 7.4), and the indicated
concentration of lauric acid ([1-14C]-lauric acid,
usually added as an aqueous 10 mM solution of
sodium laurate) in a final volume of 0.25
ml. b5 was included because it stimulates the
catalytic activity (4). Following temperature
equilibration to 37 °C for 5 min, reactions were
initiated by the addition of an NADPH-
regenerating system consisting of 0.5 mM NADP+,
10 mM glucose 6-phosphate, and 1 IU ml–1 yeast
Enzymes—Human P450 4A11 (wild-type
and F434S, C52S, C85S, C199S, C255S, and
C512S mutants) was expressed and purified as
described previously (4,63). Escherichia coli
recombinant rat NADPH-P450 reductase and
human liver cytochrome b5 (b5) were prepared as
described by Hanna et al. (64) and Guengerich
(65), respectively.
glucose
6-phosphate
dehydrogenase
(66).
Reactions generally proceeded at 37 °C for 2 min
and were terminated with 1.0 ml of ethyl acetate
containing 0.1% CH3CO2H (v/v) and, following
mixing with a vortex device, the mixtures were
centrifuged (103 ´ g for 10 min). A 0.8 ml aliquot
of the ethyl acetate layer (upper phase) was
transferred to a clean tube, and the solvent was
removed under an N2 stream.
Tissue Samples— All experiments using
mice were conducted with approved protocols by
the Institutional Animal Care and Use Committee
of Vanderbilt University and in accordance with
the NIH Guide for the Care and Use of Laboratory
animals. 129/Sv mice carrying one copy of the
human cytochrome P450 4A11 gene (CYP4A11)
(under control of its native promoter were
generated as previously described (21)) were
provided normal chow diet (Purina Laboratory
Rodent 5001; St. Louis, MO) with free access to
water and were housed in an Association for the
Assessment and Accreditation of Laboratory
The dried extracts were dissolved in 200
µl of 1:1 mixture of H2O:CH3CN containing 0.1%
CH3CO2H
(v/v)
and
10
µM butylated
hydroxytoluene, and aliquots were analyzed on a
reversed-phase (octadecylsilane, C18) HPLC
column (5 µm, 2.1 mm × 100 mm (Waters,
Milford, MA)) coupled with a radioactivity
detector (IN/US Systems β-RAM, Tampa, FL).
Reaction products and substrate were eluted at a
flow rate of 0.6 ml min–1 using an increasing linear
gradient of CH3CN (including 0.1% (v/v) HCO2H)
from 35 to 95% (v/v) over 30 min.
Animal
Care
(AAALAC)-accredited,
temperature-controlled facility with a 12 h light–
dark cycle. All studies were conducted in mice
aged 6-28 weeks of age. CYP4A11 transgenic
mice were crossed with pure Sv129 wild type
mice and offspring were genotyped for the
presence of a single copy of the human CYP411
gene as previously described (21). Organs were
collected from male transgenic mice immediately
after sacrifice and used for microsomal
preparations as described below.
Assays with P450s other than P450 4A11
were performed as described previously, with the
modification of either preincubation with DTT (1
mM) for 10 min or not (the DTT remained in the
reactions): P450 2C8– paclitaxel as substrate (67),
P450 2C9—tolbutamide as substrate (68), P450
2D6—bufuralol as substrate (69), P450 3A4—
nifedipine as substrate (70), P450 19A1—
testosterone as substrate (71), P450 21A2—
progesterone as substrate (72), P450 2A6—
coumarin as substrate (73), and P450s 1B1, 1A1,
and 1A2–7-ethoxyresorufin as substrate (74).
Site-Directed Mutagenesis—P450 4A11
was subcloned into a pBlueScript vector to
preform site-directed mutagenesis. Residues from
each site were converted to serine by using the
QuikChange II Site-Directed Mutagenesis Kit
(Agilent Technologies). The resulting plasmids
were sequenced to confirm successful mutation
and subcloned into a pCW expression vector.
Enzymatic Assays—Assays were done as
described previously (4) with minor changes.
Typical incubations included 0.2 µM P450 4A11,
0.4 µM NADPH-P450 reductase, 0.4 µM b5, 150
µM L-α-dilauroyl-sn-glycero-3-phosphocholine
Protein Oxidation—Purified recombinant
P450 4A11 (0.4 ml of a 10 µM stock solution,
stored at -80 °C in 50 mM potassium phosphate
buffer (pH 7.4) containing 20% glycerol, 1 mM
DTT, and 0.1 mM EDTA) was thawed on ice and
7

P450 4A11 Sulfenic Acid and Inhibition
reduced for 30 minutes by the addition of 1 mM
TCEP at 4 °C. A Zeba Spin Desalting Column
(Thermo) pre-equilibrated with “oxidation buffer”
(100 mM potassium phosphate buffer (pH 7.4)
containing 0.1 mM EDTA and sparged with Ar)
was used to remove reducing agents. Reduced
protein was then diluted to a concentration of 500
nM using oxidation buffer. Aliquots were treated
with varying amounts of H2O2 or TCEP. Aliquots
for the activity assay (175 pmol, described above)
were incubated with H2O2 or TCEP for 15 minutes
at 37 °C. Human erythrocyte catalase (10 units,
Sigma, catalog #C3556) was added and
incubations proceeded at 23 °C for 5 minutes to
remove H2O2. These samples were used to
measure lauric acid w-hydroxylation activity as
described above.
(from above, 100 pmol) were incubated with H2O2
or TCEP for 15 minutes. Human erythrocyte
catalase (10 units) was added and incubations
proceeded at 23 °C for 5 minutes to remove H2O2.
Trichloroacetic acid was then added to a final
concentration of 10% (v/v) and the samples were
incubated on ice for 15 minutes. The enzymes in
the samples were pelleted by centrifugation
(12,000 ´ g, 15 min, 4 °C). The supernatant was
removed from each sample, and the pellet was
washed with ice cold CH3CN. The pellet was
resuspended in 50 µl of 100 mM HEPPS buffer
(pH 8.0) containing 2% (w/v) SDS and 10 mM d5-
iodoacetanilide, and alkylation proceeded for 30
minutes at 23 °C, with shaking in the dark. Protein
was precipitated again using the same method as
above to remove heavy reagent. The pellet was
resuspended in 20 µl of 100 mM HEPPS buffer
(pH 8.0) containing 2% (w/v) SDS and 1 mM
TCEP, and reduction proceeded for 30 minutes at
50 °C. d0-Iodoacetanilide (100 mM, in acetone)
was added to a final concentration of 10 mM, and
incubation was done at 23 °C in the dark for 30
minutes, with shaking. Samples were subjected to
SDS-PAGE (10% Bis-Tris, NuPAGE, Invitrogen)
separation and stained with SimplyBlue SafeStain
(Invitrogen). The Mr region corresponding to P450
4A11 was excised, digested with trypsin (8 ng/µl)
for 16 hours in 25 mM NH4HCO3 (pH 7.8) at 37
°C and subjected to LC-MS/MS analysis.
Isotope-Coded Dimedone/Iododimedone
(ICDID) Labeling of Recombinant P450 4A11—
Additional aliquots of oxidized protein (from
above, 100 pmol) were incubated with 5 mM d6-
dimedone (from a 50 mM stock suspended in 100
mM
sodium
hydroxyethylpiperazinepropanesulfonate (HEPPS)
(pH 8.0) containing 5% NaCl (w/v)) for 2 hours at
37 °C. Trichloroacetic acid was then added to a
final concentration of 10% (v/v) and the samples
were incubated on ice for 15 minutes. The
enzymes in the samples were pelleted by
centrifugation (12,000 ´ g, 15 min, 4 °C). The
supernatant was removed from each sample, and
the pellet was washed with ice cold CH3CN. The
pellet was resuspended in 20 µl of 100 mM
HEPPS buffer (pH 8.0) containing 2% (w/v) SDS
and 1 mM TCEP, and reduction proceeded for 30
minutes at 37 °C. (d0-) Iododimedone (100 mM
Preparation of Microsomes—Microsomes
from freshly excised tissues were prepared with
slight modifications of published methods (66).
For ICDID labeling studies, the buffer (0.10 M
Tris-acetate (pH 7.4) containing 0.10 M KCl, 1.0
mM EDTA, and 20 µM BHT was sparged with Ar
before use. Tissue samples were homogenized
using a Teflon-glass Potter-Elvehjem device and
centrifuged at 104 ´ g for 20 min. at 4 °C.
Homogenates were then treated as in (66), and the
final microsomal pellets were resuspended in 100
mM HEPPS buffer (pH 8.0) containing 1 mM
EDTA. Aliquots of these samples were incubated
with vehicle, 10 mM d6-dimedone, or 10 mM d6-
dimedone plus 500 µM H2O2 for 1 hour at 37 °C.
Catalase (10 units) was added to each and
incubated at 25 °C for 10 minutes to remove
excess H2O2. Aliquots were reduced with 1 mM
TCEP at 37 °C for 30 minutes and 10 mM (d0-)
iododimedone was then added. The protein
stock, in acetone) was added to
a
final
concentration of 10 mM, and incubation was done
at 23 °C in the dark for 30 minutes. Samples were
subjected to SDS-PAGE (10% Bis-Tris, NuPAGE,
Invitrogen) separation and stained with
SimplyBlue SafeStain (Invitrogen). The Mr region
corresponding to P450 4A11 was excised, digested
with trypsin (8 ng/µl) for 16 hours in 25 mM
NH4HCO3 (pH 7.8) at 37 °C and subjected to LC-
MS/MS analysis.
Isotope-Coded Affinity Tag (ICAT)
Labeling of Recombinant P450 4A11— Additional
aliquots of reduced, buffer-exchanged protein
8

P450 4A11 Sulfenic Acid and Inhibition
concentrations of microsomes were estimated
using a bicinchoninic acid (BCA) assay (Pierce).
Alkylated microsomes (60 µg protein) were
database (Version 20160620). Precursor ion mass
tolerance was set at 10 ppm and the fragmentation
tolerance was 20 ppm for the database search.
Methionine oxidation (15.9949ꢀDa, dynamic) and
cysteine modifications by d6-and d0-dimedone
(144.1057 and 138.0681 Da, respectively, static)
were searched separately as modifications. The
maximum Q values of peptide spectrum matches
were adjusted to achieve either a peptide or a
protein false discovery rate ≤5%, using IDPicker
software (Version 3.1.642.0) (77). A spectral
library of peptides was then created with IDPicker
and loaded into Skyline Software for confident
identification of precursors pertaining to cysteine-
containing peptides. MS1 precursor quantitation
was performed as described previously (31). For
each peptide analyzed, integral areas for M and
M+1 isotopes were used for relative quantitation.
subjected
to
SDS-polyacrylamide
gel
electrophoresis (10% gel, vide supra), and the
P450 region (Mr 45-60 kDa) was excised, digested
with trypsin (8 ng/µl) for 16 hours in 25 mM
NH4HCO3 (pH 8.0) at 37 °C, and subjected to
LC/MS/MS analysis as described below.
LC-MS/MS Analysis—Extracted peptides
were analyzed on a nanoLC Ultra system
(Eksigent Technologies, Dublin, CA) interfaced
with a LTQ Orbitrap XL mass spectrometer
(Thermo Scientific, San Jose, CA). Approximately
5 pmol of peptides was reconstituted in 0.1%
HCO2H (v/v) and pressure-loaded (1.5 µl min-1)
onto a 360ꢀµm outer diameter × 100ꢀµm inner
diameter microcapillary analytical column packed
with Jupiter octadecylsilane (C18) (3ꢀµm, 300ꢀÅ,
Phenomenex) and equipped with an integrated
electrospray emitter tip. Peptides were then
separated with a linear gradient formed with 0.1%
HCO2H in H2O (solvent A) and 0.1% HCO2H in
CH3CN (solvent B) (all v/v) by increasing from 2-
45% B (v/v) over a period of 0-45 minutes at a
flow rate of 500 nl min-1. The spray voltage was
set to 2.0ꢀkV and the heated capillary temperature
to 200ꢀ°C. Higher energy collisional dissociation
(HCD) MS/MS spectra were recorded in the data-
dependent mode using a Top 2 method with an
inclusion list containing m/z values corresponding
to P450 4A11 tryptic peptides, which included
both d6- and d0-dimedonylated cysteines,
determined in silico with Skyline software (75).
MS1 spectra were measured with a resolution of
70,000, an AGC target of 1e6, and a mass range
from m/z 300 to 1,500. HCD MS/MS spectra were
acquired with a resolution of 7,500, an AGC target
of 1e5, and a normalized collision energy of 35.
Peptide m/z values that triggered MS/MS scans
were dynamically excluded from further MS/MS
scans for 20ꢀs, with a repeat count of 1.
Spectroscopy—P450 4A11 was oxidized
with H2O2 as above but the procedure was adapted
slightly for spectroscopic assays. The enzyme was
diluted with oxidation buffer to 1 µM prior to
introduction of 1 mM TCEP or 500 µM H2O2.
After oxidation, 30 units catalase was added and
the sample was incubated at 23 °C for five
minutes. To this solution, final concentrations of 1
µM NADPH-P450 reductase, 150 µM DLPC, and
0.1 unit/ml protocatechuate dioxygenase (Sigma)
were added to an anaerobic cuvette, with NADPH
(300 nmol, aqueous) in a side-arm of the cuvette.
Samples were degassed, 20 µM 3,4-
dihydroxybenzoate
(Sigma,
substrate
for
protocatechuate dioxygenase) was added to
remove oxygen (78), and samples were further
degassed using a manifold attached to both
vacuum and purified Ar (79,80), and placed under
an anaerobic CO atmosphere. The valves of the
cuvettes were sealed and multiple UV-visible
absorbance spectra were recorded using an
OLIS/Hewlett Packard 8452 diode array
spectrophotometer (On-Line Instrument Systems,
Bogart, GA). Spectra were collected from 380 to
600 nm before and after the addition of
NADPH/sodium dithionite.
Peptide Data Analysis—Raw data files
were analyzed using MyriMatch software (76)
against a decoy protein database consisting of a
forward and reversed human Uniprot/Swissprot
9

P450 4A11 Sulfenic Acid and Inhibition
Acknowledgements: We thank the Vanderbilt Proteomics Core for running some of the LC-MS/MS
samples and K. Trisler for assistance in preparation of the manuscript.
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P450 4A11 Sulfenic Acid and Inhibition
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14

P450 4A11 Sulfenic Acid and Inhibition
FOOTNOTES
Conflict of Interest: The authors report that they have no conflicts of interest with the contents of this
article. The content is solely the responsibility of the authors and does not necessarily represent the
official views of the National Institute of Health.
Author Contributions: M.E.A. synthesized d0- and d6-iodoacetanilide and did the dimedone and
iodoacetanilide labeling experiments, LC-MS assays, spectroscopic anaerobic reduction assays, and
assays of catalytic activity and analyzed results. D.K. did the site-directed mutagenesis studies. F.P.G.
synthesized d6-dimedone and (d0-) iododimedone. L.D.N., D.K., and F.P.G. did the catalytic assays with
human P450s (other than P450 4A11). C.-H.Y. did the preliminary thiol activation assays. U.S., E. F. J.,
and A.P. developed and raised the transgenic P450 4A11 mice. M.E.A. and F.P.G. wrote most of the
manuscript. F.P.G. oversaw the project. All authors contributed to the conclusions.
The abbreviations used are: b5, cytochrome b5; DLPC, L-a-1,2-dilauoryl-sn-glycero-3-phosphocholine;
DTT,
dithiothreitol;
HCD,
higher
energy
collisional
dissociation;
HEPPS,
hydroxyethylpiperazinepropanesulfonate; 20-HETE, 20-hydroxyeicosatetraenoic acid; HRMS, high
resolution mas spectrometry; ICAT, isotope-coded affinity tagging; ICDID, isotope-coded
dimedone/iododimedone; LC-MS/MS, combined liquid chromatography–mass spectrometry; P450 (or
CYP), cytochrome P450; ROS, reactive oxygen species; TCEP, tris(carboxyethyl)phosphine.
Grant Support—This work was supported in part by National Institute of Health grants R35 CA090426
(FPG), R01 GM118122 (FPG), R01 DK095761 (AP), R01 CA162433 (AP), R01 HD004445 (EFJ), and
T32 ES007028 (MEA), the Veterans Administration (I01 BX002025-01) (AP), and National
Research Foundation of Korea (Grant NRF-2016R1A2B4006978), Republic of Korea (C.-H. Y.). MEA
was also supported by American Heart Association Predoctoral Fellowship #PRE33410007. AP is the
recipient of a Veterans Administration Senior Research Career Scientist award. Some of the mass
spectrometry work was funded in part by National Institute of Health grant P30 CA 068485 Vanderbilt-
Ingram Cancer Center Support Grant.
SThe on-line version of this article (available at http://www.jbc.org) contains Supplemental Data.
15

P450 4A11 Sulfenic Acid and Inhibition
Table 1. Effects of DTT on catalytic activities of P450 4A11 and other human P450s
Catalytic activity,
nmol product/min/nmol P450
Human
P450
Substrate
Reaction
12-OH
-DTT
+DTT (1 mM)
38 ± 1
% change
+ 420
+ 7
4A11
Lauric acid
Paclitaxel
9.0 ± 1.1
1.8 ± 0.1
1.1 ± 0.1
2.9 ± 0.1
2.0 ± 0.6
2C8
6-OH
1.9 ± 0.1
1.5 ± 0.2
3.2 ± 0.1
1.8 ± 0.5
10.9 ± 0.9
2C9
Tolbutamide
Bufuralol
1´-OH
+ 33
+ 9
2D6
3A4
19A1
21A2
2A6
1A1
1A2
1B1
4´-OH
Nifedipine
Desaturation
- 10
Testosterone
Progesterone
Coumarin
Estrone formation 10.8 ± 0.2
+ 1
21-OH
51 ± 10
68 ± 6
+ 30
- 14
a
a
6-OH
a
a
a
a
a
a
Ethoxyresorufin
Ethoxyresorufin
Ethoxyresorufin
(resorufin)
(resorufin)
(resorufin)
+ 8
+ 26
- 4
Position of hydroxylation (OH) or product indicated.
aAssayed using fluorescence methods but absolute amounts of product not calibrated.
16

P450 4A11 Sulfenic Acid and Inhibition
FFiigg..1X
40
40
A
B
30
30
20
20
10
10
0
0
1
1
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
5
2
5
5
2
5
2
10 50
10 50
10 50
10 50
100
100
100
100
[DTT], μM
[DTT], μM
[TCEP], μM
[TCEP], μM
FIGURE 1.
Stimulation of lauric acid -hydroxylation activity by DTT and TCEP. P450 4A11
w
was pre-incubated with varying concentrations of either (A) DTT or (B) TCEP and rates of lauric acid w-
hydroxylation were measured.
Fig. 2
10
A
+DTT (1 mM)
8
6
wild-type
4
no DTT
2
0
0
50 100 150 200 250 300 350 400
8
6
4
2
0
+DTT (1 mM)
B
F434S
no DTT
0
50 100 150 200 250 300 350 400
[Lauric acid], μM
Figure 2.
Stimulation of lauric acid
w
-hydroxylation activity by DTT. A, wild-type P450 4A11
(CYP4A11*1); B, F434S variant (rs1126742).
17

P450 4A11 Sulfenic Acid and Inhibition
Fig. 3
100
80
60
40
20
0
100
80
60
40
20
0
0
5
10
15
Time, min
FIGURE 3.
Kinetics of loss of lauric acid
w
-hydroxylation activity of P450 4A11 in the presence
of H2O2. P450 4A11 (500 nM) was incubated with 500 µM H2O2 in “oxidation buffer” (see Experimental
Procedures) (pH 8.0) for the indicated times. Residual H2O2 was removed by treatment with human
erythrocyte catalase (50 units ml-1) for 10 minutes, and lauric acid w-hydroxylation activity was measured
in a reconstituted enzyme system. The initial (uninhibited) rate was 26.9 nmol w-hydroxylation activity
lauric acid formed min-1 (nmol P450)-1. Results are presented as means ± SD of triplicate experiments.
18

P450 4A11 Sulfenic Acid and Inhibition
Fig. 4
25
20
IC50 = 136 ± 34 µM
15
10
5
0
1
2
3
log10 [H2O2], µM
FIGURE 4.
Loss of P450 4A11 lauric acid
w
-hydroxylation activity as a function of H2O2. P450
4A11 (500 nM) was incubated with varying concentrations of H2O2 for 15 min at 37 °C in oxidation
buffer (pH 8.0). Catalase (10 units ml-1) was added to quench the residual H2O2 (for 10 min at 25 °C), and
the remaining P450 4A11 was reconstituted and used to measure lauric acid w-hydroxylation activity.
Results are presented as means ± SD of triplicate experiments. The outer lines indicate 95% confidence
intervals.
19

P450 4A11 Sulfenic Acid and Inhibition
Fig. 5
10
8
10
8
+DTT
A
B
+DTT
6
6
no DTT
4
4
no DTT
2
2
C53S
C86S
0
0
0
50 100 150 200 250 300 350 400
0
50 100 150 200 250 300 350 400
10
8
10
8
+DTT
+DTT
D
C
6
6
no DTT
C256S
4
4
2
2
no DTT
C200S
0
0
0
50 100 150 200 250 300 350 400
0
50 100 150 200 250 300 350 400
10
E
8
6
4
2
0
+DTT
no DTT
C513S
0
50 100 150 200 250 300 350 400
[Lauric acid,], µM
FIGURE 5.
Lauric acid -hydroxylation activity of Cys Ser mutants of P450 4A11. For
w ®
experiments done at the same time with wild-type P450 4A11, see Fig. 2A. Assays were done in the
absence (<) and presence (=) of 1 mM DTT, as indicated. A, C43S; B, C86S; C, C200S; D, C255S; E,
C513S.
20

P450 4A11 Sulfenic Acid and Inhibition
Fig. 6
SH
SH
HS
HS
HS
Remove reductant
Add H2O2
S
S
HS
HS
HS
HS
SH
HOS
HS
HS
D
D
D
D
D
O
O
I
CD3
CD3
N
H
Activity assay
d5-Iodoacetanilide
D5IAA
S
O
(IAA)
d6-Dimedone
S
(DMD)
S
D6DMD
HS
SH
S
S
S
D5IAA
D5IAA
HS
HS
O
O
CH3
CH3
I
I
N
H
O
d0-Iodoacetanilide
D0IAA
D0IAA
d0-Iododimedone
D5IAA
D0DMD
D0DMD
S
D6DMD
S
S
S
S
S
S
S
D5IAA
S
S
D5IAA
D0DMD
D0DMD
FIGURE 6.
Scheme for analysis of sulfenic acids and disulfides of P450 4A11.
21

P450 4A11 Sulfenic Acid and Inhibition
Fig. 7
204.1337
y
2
100
A
173.0628
N C* I G K
y
y
y
y
4
3
2 1
50
317.2168
y
3
558.2952
147.1124
y
y
4
1
0
100
200
300
400
m/z
500
600
204.1331
y
2
336.6719
2.7 ppm
100
100
B
339.6906
3.2 ppm
340.1931
179.0998
337.1737
339.1876
339.0
340.6957
340.0 341.0
337.6700
338.0
0
337.0
50
m/z
317.2170
y
3
147.1123
y
1
564.3211
y
4
0
100
200
300
400
m/z
500
600
FIGURE 7.
Peptide analysis. Annotated HCD MS/MS of the heme-thiolate containing peptide (456-
NCIGK-460) alkylated with d0-dimedone (A) or d6-dimedone (B).
22

P450 4A11 Sulfenic Acid and Inhibition
Fig. 8
Cys-457-SH
NCIGK - 33C9.6y91s7-+4+5(h7ea-vSy)OH
NCIGK - 336.6729++
880000
700
600
500
440000
300
200
100
00
DMD_TCEP
DMD_5uM
20160901_DMD_100uM
20160901_DMD_500uM
Reduced
5 μM H O
100 μM H O
500 μM H O
2
2
2
2
2
2
Replicate
70
50
40
30
20
10
0
140
120
100
80
60
50
40
30
20
10
0
39.5
38.5
38.8
-0.5 ppm
-3.3 ppm
-2.7 ppm
38.7
60
50
40
30
-2.1 ppm
38.8
-2.4 ppm
38.6
-2.2 ppm
60
38.6
20
10
40
-2.5 ppm
39.4
20
-0.6 ppm
0
0
38.2 38.4 38.6 38.8 39.0 39.2
38.0
38.2
38.4
38.6
38.8 39.0
39.2
38.2
38.4
38.6
38.8
39.0
39.2
39.0
39.2
39.4
39.6
39.8
40.0
Retention Time
Formation of a sulfenic acid in Cys-457 (heme thiol group) as a function of H2O2
FIGURE 8.
concentration. P450 4A11 samples were labeled with d6-dimedone and and counter-alkylated with d0-
iododimedone at varying concentrations of H2O2. Labeled protein was then subject to trypsin digestion
and LC/MS/MS analysis. Masses corresponding to Cys-457 deuterated and non-deuterated peptides were
extracted and the resulting areas were integrated using Skyline software. A, bar graphs of comparisons of
peak areas for CySH peptide (red) and CyS-OH peptide (blue); raw peaks for CySH peptide (red) and
CyS-OH peptide (blue) (not normalized in each case).
23

P450 4A11 Sulfenic Acid and Inhibition
Fig. 9
0.15
0.10
0.05
Reductase
A
Dithionite
400
500
600
420
450
B
Reductase
Dithionite
0.15
0.10
0.05
400
500
550
420
450
Wavelength, nm
FIGURE 9.
Reduction of P450 4A11 by NADPH-P450 reductase and sodium dithionite in the
presence of CO. Reduced protein (A) and protein oxidized with 500 µM H2O2 (B) was deaerated and
placed under an anaerobic CO atmosphere. NADPH was then added and absorbance spectra was recorded
(blue). Dithionite was then added (red).
24

P450 4A11 Sulfenic Acid and Inhibition
Fig. 10
P420
P450
CO
CO
FeII
NADPH-P450
reductase
H2O2
Dithionite
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
FeIII
FeIII
FeII
(Na2S2O4)
CO
OH
OH
S
S
S
S
4A11
4A11
4A11
4A11
FIGURE 10.
Scheme of Cys-457 sulfenic acid formation and reduction in P450 4A11. In the
presence of H2O2, the heme-thiolate ligand becomes oxidized and loses its coordination with the heme
iron. The sulfenic acid is not reduced by NADPH-P450 reductase but can be reduced by dithionite,
reforming the heme coordination.
25

P450 4A11 Sulfenic Acid and Inhibition
Fig. 11
100
80
60
40
20
0
100
80
60
40
20
0
Kidney 4A11
Liver 4A11
A
B
D
2
2
O
O
2
2
Control
Control
Dimedone
Dimedone
Dimedone + H
Dimedone + H
100
80
60
40
20
0
80 C
Liver 4A12
60
40
20
0
Kidney 4a12
2
2
O
O
2
2
Control
Control
Dimedone
Dimedone
Dimedone + H
Dimedone + H
100
80
60
40
20
0
Kidney 4b1
E
2
O
2
Control
Dimedone
Dimedone + H
26

P450 4A11 Sulfenic Acid and Inhibition
FIGURE 11.
Analysis of human and mouse P450 peptides in tg4A11 mice. Microsomes (prepared
from kidney and liver tissues of CYP4A11 transgenic mice within 3 hours after sacrifice) were treated
with either vehicle (control), d6-dimedone, or d6-dimedone supplemented with 500 µM H2O2. Integrated
areas for ions corresponding to peptides of the heme-thiolate for P450s 4A11 (A, B), 4a12 (C, D), and 4b1
(E) were compared and reported as percent sulfenylation (percent sulfenylation = area of d6-dimedone-
labeled peptide/(area of d6-dimedone-labeled peptide plus area of d0-dimedone-labeled peptide)). See
Experimental Procedures and Fig. 6. The p values using a Students t-test comparing control- and
dimedone-treated tissue were all p <0.03 except for the P450 4a12 kidney (C) (p = 0.058). The mouse
indicated with the label “q“ was 6 weeks old, much younger than the other three animals (two were 3
months old and one was 7 months old).
27

P450 4A11 Sulfenic Acid and Inhibition
A
B
FIGURE 12.
Homology model of P450 4A11 based on crystal structure of rabbit P450 4B1 (57).
A, All Cys residues are shown; B, positions of Asn-456, Lys-460, and Phe-462 near the heme and Cys-
457 (yellow sphere).
28

Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox
switch for catalytic inhibition
Matthew E. Albertolle, Donghak Kim, Leslie D. Nagy, Chul-Ho Yun, Ambra Pozzi, Uzen
Savas, Eric F. Johnson and F. Peter Guengerich
J. Biol. Chem. published online May 22, 2017
Access the most updated version of this article at doi: 10.1074/jbc.M117.792200
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