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

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 Kim^1,2, Leslie D. Nagy¹, Chul-Ho Yun³, Ambra Pozzi^4,5, 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

H₂O₂attenuated 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

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 k_catvalues for both the wild-type

and F434S (rs1126742) (8) variants of P450 4A11

(2.1-fold) (Figs. 2A, 2B). The K_mvalues 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). H₂O₂modifies epidermal growth

factor receptor signaling (23) and regulates

glycolysis through glyceraldehyde-3-phosphate

dehydrogenase (24). Common targets of H₂O₂are

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% d₀labeling) 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 A₄₅₀upon 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 H₂O₂was

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 H₂O₂and 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 H₂O₂required 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 H₂O₂in

conjunction with NADPH-P450 reductase and

NADPH, with or without substrate present (43).

H₂O₂production 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 H₂O₂produced 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 H₂O₂produced 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 H₂O₂led 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 H₂O₂reacts 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 (-SO₂and -

–

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

λ_maxat 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 λ_maxat 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 C₈H₇D₆O₂m/z

In summary, P450 4A11 seems to show a

unique activation under reducing conditions

compared to other P450 enzymes tested.

Conversely, H₂O₂inhibits 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. b₅was 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^–1yeast

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 N₂stream.

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^–1using 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 H₂O₂or TCEP. Aliquots

for the activity assay (175 pmol, described above)

were incubated with H₂O₂or 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 M_rregion 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 H₂O₂for 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 M_rregion

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. MS¹precursor 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 (M_r45-60 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 H₂O₂as 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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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

6-OH

a

Ethoxyresorufin

(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

A

B

30

20

10

0

1

0

1

0

2

5

2

5

2

5

2

10 50

100

[DTT], 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

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

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 H₂O₂in “oxidation buffer” (see Experimental

Procedures) (pH 8.0) for the indicated times. Residual H₂O₂was 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 H₂O₂for 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

no DTT

4

no DTT

2

C53S

C86S

0

50 100 150 200 250 300 350 400

0

50 100 150 200 250 300 350 400

10

8

10

8

+DTT

D

C

6

no DTT

C256S

4

2

no DTT

C200S

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

HS

Remove reductant

Add H₂O₂

S

HS

SH

HOS

HS

D

O

I

CD₃

N

H

Activity assay

d₅-Iodoacetanilide

D₅IAA

S

O

(IAA)

d₆-Dimedone

S

(DMD)

S

D₆DMD

HS

SH

S

D₅IAA

HS

O

CH₃

I

N

H

O

d₀-Iodoacetanilide

D₀IAA

d₀-Iododimedone

D₅IAA

D₀DMD

S

D₆DMD

S

D₅IAA

S

D₅IAA

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

4

3

2 1

50

317.2168

y

3

558.2952

147.1124

y

4

1

0

100

200

300

400

m/z

500

600

204.1331

y

2

336.6719

2.7 ppm

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 - 33}C_9.6y₉₁s₇-₊4₊5_(h7_ea-_vS_y)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

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

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

Fe^II

NADPH-P450

reductase

H₂O₂

Dithionite

N

Fe^III

Fe^II

(Na₂S₂O₄)

CO

OH

S

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

O

2

Control

Dimedone

Dimedone + H

100

80

60

40

20

0

⁸⁰C

Liver 4A12

60

40

20

0

Kidney 4a12

2

O

2

Control

Dimedone

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

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

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