THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 7, pp. 4755–4762, February 15, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
RegB Kinase Activity Is Repressed by Oxidative Formation of
Cysteine Sulfenic Acid*
Received for publication, August 24, 2012, and in revised form, January 9, 2013 Published, JBC Papers in Press, January 10, 2013, DOI 10.1074/jbc.M112.413492
Jiang Wu‡, Zhuo Cheng‡, Khalilah Reddie§, Kate Carroll¶, Loubna A. Hammad储, Jonathan A. Karty储,
and Carl E. Bauer‡1
From the ‡Department of Molecular and Cellular Biochemistry and the 储METACyt Biochemical Analysis Center, Department of
Chemistry, Indiana University, Bloomington, Indiana 47405, the §Life Sciences Institute, University of Michigan, Ann Arbor,
Michigan 48109, and the ¶Department of Chemistry, The Scripps Research Institute, Florida Campus, Jupiter, Florida 33458
RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense
environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to
form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive
tetramer. In this study, we demonstrate that a stable sulfenic
acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo
when RegB is exposed to oxygen. This sulfenic acid modification
is reversible and stable in the air. Autophosphorylation assay
shows that reduction of the SOH at Cys-265 to a free thiol (SH)
can increase RegB kinase activity in vitro. Our results suggest
that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of
Cys-265 under aerobic conditions. Cys-265 thus functions as a
complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase
activity of RegB.
The RegB/RegA two-component system was initially discovered in the purple non-sulfur photosynthetic bacterium Rhodobacter capsulatus as a key regulator of photosystem synthesis
(1, 2). RegB is a membrane-associated histidine kinase that
autophosphorylates under reducing conditions then transfers
the phosphoryl group to RegA (3, 4). The phosphorylation state
of RegA subsequently affects gene expression by affecting how
RegA interacts with the promoter and RNA polymerase (4).
The RegB/RegA regulon is quite extensive and is known to
include genes involved in synthesis of the photosystem, respiration, electron transport, carbon fixation, nitrogen fixation,
hydrogen oxidation, heme biosynthesis, and aerotaxis (5).
Highly conserved homologous systems are also found in many
* This work was supported, in whole or in part, by National Institutes of Health
Grant R37 GM040941 (to C. E. B.) and Grant R01 GM102187 (to K. S. C.) as
well as funding from the Camile Henry Dreyfus Teacher Scholar Award.
1
To whom correspondence should be addressed: Department of Molecular
and Cellular Biochemistry, Simon Hall MSB, 212 S. Hawthorne Dr., Indiana
University, Bloomington, IN 47405. Tel.: 812-855-6595; Fax: 812-856-5710;
E-mail: bauer@indiana.edu.
FEBRUARY 15, 2013 • VOLUME 288 • NUMBER 7
other photosynthetic and non-photosynthetic proteobacteria
where they control many energy-generating and energy-utilizing biological processes in response to changes in cellular and
environmental redox (6). The redox-sensing mechanism by
which RegB perceives changes in environmental oxygen tension has been extensively studied but several aspects of its control remain unanswered.
There are three models proposed to explain the redox regulatory mechanism of RegB. The first model suggests that a
“redox-active” cysteine Cys-265 in the RegB cytosolic domain is
a redox sensor that is capable of forming an intermolecular
disulfide bond under oxidizing conditions to convert an active
RegB dimer into an inactive RegB tetramer (7). However, Western blot analysis showed that only about 20% of RegB dimers
were cross-linked under aerobic growth conditions, suggesting
that additional regulation occurred beyond that of disulfide
bond formation. A second model is centered on a highly conserved ubiquinone-binding site located in the periplasmic loop
between transmembrane helices 3 and 4. Mutational studies
suggest that ubiquinone binding to this site acts as an input
signal that controls kinase activity in response to the redox state
of the ubiquinone pool (8, 9). The fact that a RegB C265S
mutant maintains the ability to respond to redox changes in
ubiquinone also indicates that the ubiquinone-binding site and
Cys-265 function independently from each other and that they
sense different signals (8). The last model is based on studies
from PrrB (a homologue of RegB in Rhodobacter sphaeroides)
that showed that mutations in cbb3 cytochrome c oxidase result
in increased aerobic PrrB/PrrA-regulated gene expression. It
has been proposed that the cbb3 cytochrome c oxidase is the
redox sensor that generates an inhibitory signal under aerobic
conditions to shift PrrB from a kinase-dominant mode into a
phosphatase mode (10). However the exact identity of the
inhibitory signal and a detailed mechanism has not been specified, so it cannot be ruled out that mutations in cytochrome c
oxidase simply affect the redox state of the ubiquinone pool,
which subsequently affects RegB activity.
In this study we further analyze the role of the cytosolic
located Cys-265 as a redox switch that controls the activity of
RegB. As discussed above, only ⬃20% of RegB form disulfideJOURNAL OF BIOLOGICAL CHEMISTRY
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Background: Activity of the redox responsive global sensor kinase RegB is repressed under aerobic conditions.
Results: Stable and reversible sulfenic acid modification forms at cysteine 265 to repress kinase activity.
Conclusion: Cysteine 265 functions as a redox switch that regulates kinase activity in response to aerobic conditions.
Significance: This study discloses additional regulatory details of the redox-sensing sensor kinase RegB.
Cys-265 Oxidation Inhibits RegB Kinase Activity
EXPERIMENTAL PROCEDURES
Strains, Media, and Growth Conditions—R. capsulatus strain
SB1003 containing a chromosome encoded wild-type Flagtagged RegB or Flag-tagged RegB C265A were constructed as
previously described (7). pET29(⫹) plasmid encoding for the
truncated version of RegB (RegB⬙) and C265A mutant that
starts at amino acid Met-196 with an N terminus S-tag was also
constructed as previously described (7). BL21(DE3) was used
for overexpression of RegB⬙ and RegB⬙ C265A. Terrific broth
was used for BL21(DE3) cultures, and PY media was used for all
R. capsulatus strains. 25 ␮g/ml kanamycin and 1.5 ␮g/ml gentamycin were added to BL21(DE3) and R. capsulatus liquid cultures, respectively.
Overexpression and Purification of S-tagged RegB⬙—
BL21(DE3) strains carrying pET29RegB⬙ and pET29RegB”C265A
were grown in terrific broth with 25 ␮g/ml kanamycin to an
A600 of 0.4. IPTG was then added to 0.4 mM to induce overexpression at 16 °C overnight. Cells were harvested by centrifugation and suspended in 20 mM Tris-HCl, pH 8.0, 10 mM ␤-mercaptoethanol, and then lysed by three passes through a M-110L
Micro Fluidizer Processor (Microfluidics). After clarifying the
cell lysate by centrifugation at 22,000 ⫻ g for 30 min at 4 °C, the
supernatant was loaded to a Hitrap Q (HP) column (GE Health-
2
The abbreviations used are: DAz-2, 4-(3-azidopropyl)cyclohexane-1,3-dione; CID, collision-induced dissociation spectrum; LC-ESI-MS/MS, liquid
chromatography electrospray tandem ionization mass spectrometric analysis; EIC, extracted ion chromatogram; p-CMB, p-chloromercuribenzoate;
DMSO, dimethyl sulfoxide.
4756 JOURNAL OF BIOLOGICAL CHEMISTRY
care) and eluted with a 0 –500 mM NaCl gradient in 20 mM
Tris-HCl, pH 8.0, 10 mM ␤-mercaptoethanol. Purified RegB⬙
was reconstituted with Cu2⫹ by incubating with 300 ␮M CuCl2
then extensively dialyzed against 3 ⫻ 2 liters of 20 mM Tris-HCl,
pH 8.0, 300 mM NaCl (a previous study has shown that a metal
is needed to form redox active RegB⬙; Ref. 7). To separate RegB⬙
dimer and tetramer, a Sephacryl S-200 High-resolution column
was connected to a Superose 12 column to fractionate RegB⬙ in
20 mM Tris-HCl, pH 8.0, 300 mM NaCl.
In Vitro DAz-2 Labeling of Sulfenic Acid in Recombinant
RegB⬙—DAz-2 labeling of purified RegB⬙ is modified from the
method described by K. Reddie et al. (20). 2000 ng of freshly
purified RegB⬙ or RegB⬙ C265A were reacted with 0.25 mM
DAz-2 or DMSO for 1 h at 37 °C. In experiments where RegB⬙
was treated with H2O2 or air oxygen, RegB⬙ was first reduced by
10 mM DTT at 22 °C for 30 min in an anaerobic hood. After
DTT was removed by ultrafiltration with Amicon Ultra Filters
(10 kDa, Millipore), reduced RegB⬙ was then incubated with 1
equivalent H2O2 in the hood or exposed to air outside the hood
at 22 °C for 30 min. DAz-2 labeling was then carried out as
described above.
In Vivo DAz-2 Labeling of Sulfenic Acid in RegB-FLAG in
R. capsulatus Cells—Growth of aerobic cultures of R. capsulatus strain SB1003 carrying RegB-FLAG or RegB C265A-FLAG
involved incubating 250 ml of PY medium in 3-liter baffled
flasks with overnight cultures. The cultures were grown at 34 °C
until A600 reached 0.2– 0.3. Anerobic cultures were grown in PY
medium in an airtight screw-cap bottle at 30 °C under light for
about 24 h until A600 reached 1.6 –1.7.
DAz-2 labeling of RegB-FLAG in R. capsulatus cells
occurred with 250 ml of aerobic culture or 50 ml of anaerobic
culture. After reaching appropriate cell density, the cells were
chilled on ice, collected by centrifugation and washed three
time with 50 ml of cold TBS (50 mM Tris HCl, 150 mM NaCl, pH
7.4). The cell pellet was suspended in 3 ml of TBS and incubated
with 5 mM DAz-2 or DMSO at 4 °C for 3 h. The cells then were
washed three time with 50 ml cold TBS and suspended in 3 ml
of 50 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 1 mg/ml
lysozyme, 20 ␮l of protease inhibitor mixture (Sigma), pH 7.4.
The cell lysate was prepared by sonication. After centrifugation
at 20,000 ⫻ g for 20 min, the supernatant was incubated with
EZView Red ANTI-FLAG M2 affinity gel (Sigma) overnight at
4 °C to enrich the labeled RegB-FLAG. The resin was collected
at 8,200 ⫻ g for 30 s and washed three times with 0.5 ml of TBS.
DAz-2 labeled RegB-FLAG was eluted with 100 ␮l of 150 ng/␮l
3⫻ FLAG peptide (Sigma).
Biotinylation of RegB and Western Blot Analysis—DAz-2-labeled recombinant RegB or RegB-FLAG was conjugated to biotin via Staudinger ligation with 0.25 mM phosphine-biotin for
2 h at 37 °C. Biotinylation reactions were terminated by the
addition of 0.5 ml of cold acetone and kept in ⫺80 °C for 30 min.
The precipitated protein was centrifuged at 20,000 ⫻ g for 15
min, and the protein pellet was resuspended in TS buffer (50
mM Tris, 1% SDS, pH 7.4).
Biotinylated proteins in TS buffer were separated by 15%
SDS-PAGE and transferred to nitrocellulose membrane (Perkin Elmer). The membrane was blocked in 3% bovine serum
albumin (BSA) (Fisher) in phosphate-buffered saline Tween-20
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linked tetramers when exposed to oxygen in vivo. Despite this
modest oxidation, more than 95% of the kinase activity is
repressed, suggesting that signals other than disulfide bond formation also control activity (7).
It has been known that distinct modifications other than that
of a disulfide formation can occur at protein thiols in response
to oxidative stress, nitrosative stress, and changes in the overall
cellular redox (11, 12). These modifications include S-sulfenylation, S-nitrosylation, and S-glutathionylation and have been
implicated in the regulation of enzymes and transcriptional factors (13–16). Among these modifications, sulfenic acid (RSOH)
is emerging as an important redox modification involved in
signaling oxidative stress (17–19). In this study we analyzed
sulfenic acid formation in RegB in R. capsulatus cells and in
purified RegB⬙ (a truncated version of RegB that lacks the transmembrane domain) using a cell permeable sulfenic acid-specific probe 4-(3-azidopropyl)cyclohexane-1,3-dione (DAz-2).2
We found evidence that a stable sulfenic acid can form at Cys265 in vivo and in vitro. The formation of sulfenic acid is detectable in vivo upon exposure of cells to atmospheric oxygen and
undetectable in the absence of air. Sulfenic acid is also observed
with air-oxidized RegB⬙ and absent after treatment with the
reductant dithiothreitol (DTT). Autophosphorylation assays
using RegB⬙ dimers showed that specific reduction of the sulfenic acid to a free thiol increases RegB⬙ kinase activity. These
results indicate that sulfenic acid modification at Cys-265 functions as a reversible redox switch regulating RegB kinase activity in response to aerobic conditions.
Cys-265 Oxidation Inhibits RegB Kinase Activity
FEBRUARY 15, 2013 • VOLUME 288 • NUMBER 7
FIGURE 1. Non-reducing SDS-PAGE analysis of the oligomeric states of
RegBⴖ. A, oligomeric states of untreated, DTT-treated, and potassium ferricyanide-treated RegB⬙ on non-reducing SDS-PAGE. Aliquots of air-oxidized
RegB⬙ were untreated, treated with 10 mM DTT or 1 mM ferricyanide at 22 °C
for 15 min, respectively. Resultant samples were then separated by non-reducing SDS-PAGE and quantified by ImageJ. B, oligomeric state of the isolated RegB⬙ dimer on non-reducing SDS-PAGE after prolonged exposure to
atmospheric oxygen. RegB⬙ dimer was analyzed by non-reducing SDS-PAGE
right after isolation by size exclusion chromatography. After exposure to air at
4 °C for 5 days, the same sample was analyzed by non-reducing SDS-PAGE
again to observe changes in oligomeric state.
was obtained through standard purification procedure.
Reduced RegB⬙ was prepared by incubating RegB⬙ with 10 mM
DTT at 22 °C for 1 h. Reduction of sulfenic acid was carried out
by incubating RegB⬙ with 20 mM sodium arsenite at 37 °C for
1 h.
Autophosphorylation Assays—Autophosphorylation assays
were performed as described previously (4). To reduce the sulfenic acid in RegB⬙ dimer, 10 mM DTT was added to the reaction mixture and incubated at 22 °C for 15 min before 1.0 mM
ATP and 200 – 400 ␮Ci [␥-32P]ATP (7000 Ci/mmol, MP Biomedicals) were added to initiate the autophosphorylation.
RESULTS
Sulfenic Acid Formation at Cys-265 in Vitro—In a previous
study we demonstrated that aerobically grown cells have ⬃20%
of RegB in an inactive tetramer form. This is contrasted by
anaerobically grown cells where RegB is predominantly in a
dimer form (7). That study also demonstrated that aerobic
tetramerization of RegB was dependent on the presence of Cys265 suggesting that tetramerization was driven by disulfide
bond formation at Cys-265. To confirm this hypothesis we analyzed the oligomeric state of purified air-oxidized RegB⬙, a truncated version of RegB without the transmembrane domain (7),
by performing non-reducing SDS-PAGE. Consistent with the
previous in vivo study, the result of non-reducing SDS-PAGE
shows that ⬃35% of air oxidized RegB⬙ existed as tetramer that
electrophoresed at ⬃120 kDa (Fig. 1A). The remaining ⬃65% of
RegB⬙ electrophoresed at ⬃30 kDa, which is a size that represents monomers. Previous size exclusion chromatography has
shown that air-oxidized RegB⬙ is nearly exclusively a mixture of
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(PBST:1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl 2.7 mM
KCl, 0.05% Tween 20, pH 7.3) overnight at 4 °C or 1 h at 22 °C.
The membrane was then incubated with a 1:50,000 dilution of
streptavidin-HRP (GE Healthcare) in PBST for 1 h at 22 °C,
washed three times by 50 ml of TBST, and developed with
Amersham Biosciences ECL Prime Western blotting Detection
Reagents (GE Healthcare). RegB-FLAG enriched from R. capsulatus cells for in vivo analysis was identified using monoclonal
ANTI-FLAG M2-HRP (Sigma).
Mass Spectrometry—Air-oxidized RegB” (⬃40 ␮M) was first
reacted with 400 ␮M iodoacetamide in the dark at 22 °C for 45
min. 20 mM DAz-2 then was added at 22 °C for 2 h to label the
sulfenic acid. After separating treated RegB⬙ by non-reducing
SDS-PAGE, RegB⬙ monomer (corresponding to dimer in solution) and tetramer were excised from the gel and in-gel digested
with 1/20 (weight ratio) sequencing grade chymotrypsin
(Roche Applied Science) was performed overnight at 22 °C in
100 mM Tris-HCl, pH 7.9, 10 mM CaCl2. The resulting peptides
were extracted and then analyzed using the LTQ Orbitrap
instrument. The analysis was performed using an Eksigent
nano LC system (AB SCIEX, Farmingham, MA) interfaced to
an externally calibrated LTQ Orbitrap hybrid mass spectrometer equipped with a nanospray ion source (Thermo Scientific,
San Jose, CA). A 5-␮l aliquot of the peptide digest dissolved in
15 ␮l of mobile phase A was on-line washed with mobile phase
A for 6 min at a flow rate of 5 ␮l/min using an in-house packed
reversed-phase C18 (200 Å, 5 ␮m Magic C18AQ200 particles,
Michrom Bioresources, Auburn, CA) trap column (15 mm ⫻
100 ␮m, 50 ␮m frit) (New Objective, Woburn, MA). The
desalted peptides were then separated using an in-house
packed reversed-phase C18 (100 Å, 5 ␮m Magic C18AQ100
particles, Michrom Bioresources, Auburn, CA) column (150
mm ⫻ 75 ␮m, 15 ␮m tip) (New Objective, Woburn, MA) and
analyzed in positive ion mode on the LTQ-Orbitrap instrument. Mobile phase A consisted of 0.1% formic acid in acetonitrile:water (3:97%, v/v) and mobile phase B consisted of 0.1%
formic acid in acetonitrile:water (97:3%, v/v). The flow rate was
300 nL/min. A 60-minute gradient from 97% mobile phase A to
10% mobile phase A was used with a total run time of 88 min.
The mass spectrometer was operated in an automated data
dependent mode alternating between an FT-MS scan in the
Orbitrap and a collision-induced dissociation (CID) scan in the
linear ion trap. The scan in the Orbitrap was from m/z 300 to
m/z 2000 at 15,000 resolving power. The precursor ions were
isolated in the linear trap using the data-dependent acquisition
mode with a 2 m/z isolation width to select automatically and
sequentially the three most intense ions from each survey scan.
The isolated ions were collisionally activated in the linear trap
at 35% normalized collision energy. The cycle was continuously
repeated throughout the entire separation with the dynamic
exclusion set to 45 s for a repeat count of 2. The orbitrap high
resolution enabled accurate mass assignments for the precursor ions and assured the ability to determine the charge states of
peptides.
Free Thiol Quantification—Free thiols were quantified by
adding aliquots of 0.2 mM p-chloromercuribenzoate (p-CMB)
in 0.1 M Tris-HCl at pH 7.3 (21, 22) to ⬃20 ␮M RegB⬙ with
increases in absorbance at 250 nm recorded. Air oxidized RegB⬙
Cys-265 Oxidation Inhibits RegB Kinase Activity
4758 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 2. Oxidation state of Cys-265 in air-oxidized RegBⴖ observed by
LC-ESI-MS/MS. Air-oxidized RegB⬙ was first treated with iodoacetamide and
DAz-2. After separation by non-reducing SDS-PAGE, RegB⬙ monomer and
tetramer were excised from the gel and in-gel digested with chymotrypsin
with the digested peptides subsequently subjected to LC-ESI-MS/MS analysis. A, EIC, mass spectrum (inset), and CID spectrum of intermolecular Cys-265
disulfide-bonded peptide ion at m/z 750.8. The peptide sequence and fragment ion assignments are listed on the figure. B, EIC, mass spectrum (inset)
and CID spectrum of iodoacetamide-labeled (CAM) Cys-265-containing peptide ion at m/z 520.27. The peptide sequence and fragment ion assignments
are listed on the figure. C, EIC, mass spectrum (inset), and CID spectrum of the
peptide ion at m/z 424.4 containing DAz-2-labeled Cys-265. The peptide
sequence and fragment ion assignments are listed on the figure.
We also assayed whether sulfenic acid formation can occur in
response to oxygen. In this assay, RegB⬙ was reduced by 10 mM
DTT in an anaerobic hood, followed by exposure to air oxygen.
Treated samples then were labeled by DAz-2, biotinylated by
p-biotin, and then analyzed by Western blots. Results showed
that while very low level of labeling was detected in the reduced
RegB⬙, the intensity of the labeling was dramatically increased
in the RegB⬙ sample exposed to air (Fig. 3B). This indicates that
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tetramer and dimer forms with no observable monomers (7). It
was also shown that SDS can dissociate RegB⬙ dimers into
monomers (7). Consequently, the monomer band observed by
SDS-PAGE actually represents the fraction of RegB that is in a
dimer form in solution. As would be expected of disulfide-mediated oligmerization, DTT treatment disrupted nearly all of
the tetramers (Fig. 1A). However, the converse experiment of
oxidation of RegB⬙ with the strong oxidizing agent potassium
ferricyanide (K3[Fe(CN)6]) at 22 °C for 15 min only converts
⬃72% of RegB⬙ into tetramers (Fig. 1A). Given that air oxidized
RegB has nearly indetectable activity in vitro (7), the relatively
low amount of RegB tetramers observable upon exposure to
oxygen suggests that oxidative modifications other than a disulfide bond may be occurring at Cys-265.
We further analyzed oxidation events occurring at Cys-265
by undertaking liquid chromatography electrospray tandem
ionization mass spectrometric analysis (LC-ESI-MS/MS) of
RegB⬙. Air-oxidized RegB⬙ was first treated with iodoactamide,
which conjugates to free thiols, and then incubated with DAz-2
that selectively labels Cys sulfenic acid derivatives (23). Treated
RegB⬙ were then fractionated by non-reducing SDS-PAGE with
the tetramer and monomer (corresponding to dimer in solution) bands of RegB⬙ excised from the gel and digested by chymotrypsin prior to LC-ESI-MS/MS analysis. The identities of
the Cys-265-containing peptides were confirmed by high mass
accuracy and by manual interpretation of their CID) spectra
(Fig. 2, A–C).
In the digested RegB⬙ tetramer, a peak was observed at m/z
750.8926(M⫹4H)4⫹ (4 ppm mass error) that corresponds to
two copies of the chymotyptic peptide IREQAERC265RDIL
linked by a disulfide bond at Cys-265 (Fig. 2A, inset). This is
contrasted by the monomer sample that gave quite different
results. LC-MS-MS data indicated that the monomer had the
same IREQAERC265RDIL chymotryptic peptide with the difference that there is no detectable disulfide bond. Instead, there
is a peak at m/z 520.2733 (M⫹3H)3⫹ (⫺4 ppm error, Fig. 2B,
inset) corresponding to a free thiol modified by iodoacetamide
at m/z 520.2733 (M⫹3H)3⫹ (⫺4 ppm error, Fig. 2B) as well as a
peak at m/z 424.4733 (M⫹4H)4⫹ (⫺3 ppm error, Fig. 2C, inset)
corresponding to a sulfenic acid modified by DAz-2. Each of
these peaks were isolated and further characterized by CID
spectral analysis (Fig. 2). These MS results confirm the presence
of a stable sulfenic acid modification in air-oxidized RegB⬙.
To visually confirm that a sulfenic acid forms at Cys-265 in
vitro, we used the sulfenic acid-specific probe DAz-2 that contains an azide chemical handle, which allows the conjugation of
a biotin group for subsequent detection (20, 23). To detect sulfenic acid, the RegB⬙ sample was incubated with DAz-2 or
DMSO as negative control and then subjected to bio-orthogonal labeling with phosphine-biotin (p-biotin) via Staudinger
ligation (24). RegB⬙C265A mutant was also labeled in a parallel
experiment to confirm that sulfenic acid is indeed formed at
Cys-265. The labeled RegB⬙ and RegB⬙C265A were separated
by SDS-PAGE and analyzed by streptavidin-HRP Western blot.
The result clearly showed that RegB⬙ was labeled by DAz-2 and
that there is no detectable labeling in RegB⬙ C265A (Fig. 3A).
This confirms that air-oxidized RegB⬙ contains a sulfenic acid
at Cys-265.
Cys-265 Oxidation Inhibits RegB Kinase Activity
oxygen is capable of promoting the formation of sulfenic acid in
vitro and suggests molecular oxygen may be a signal for Cys-265
in vivo.
Quantification of the Sulfenic Acid at Cys-265 in RegB⬙—We
analyzed the relative abundance of sulfenic acid and other oxidation states of Cys265 by quantifying the free thiol content in
air-oxidized RegB⬙, DTT treated air-oxidized RegB⬙, and
sodium arsenite (NaAsO2)-treated air-oxidized RegB⬙. Free
thiol content was determined by measuring an increase in
absorbance at 250 nm that occurs upon mercaptide formation
between free thiols and p-chloromercuribenzoate (p-CMB)
(25, 26). The p-CMB titration results showed that the air-oxidized RegB⬙ contains ⬃0.1 free thiol per monomer (Fig. 4).
Since Cys-265 is the only cysteine in RegB⬙, it suggests most
thiols are in an oxidized state. DTT treatment of the air-oxidized RegB⬙ increased the free thiol content to ⬃0.9 free thiol
per monomer (Fig. 4), indicating most cysteine modifications
are reversible. These modifications are either disulfide bond or
sulfenic acid as both forms can be reduced to free thiol by DTT.
To determine which fraction contains sulfenic acid we treated
air oxidized RegB⬙ with the reductant NaAsO2 that can specifically reduce sulfenic acid to a free thiol (27). In this case the
NaAsO2 treated RegB⬙ sample was found to contain ⬃0.5 free
thiol per monomer (Fig. 4). The increase in the free thiol content from ⬃0.1 free thiol per monomer in air oxidized RegB⬙ to
⬃0.5 free thiol per monomer in NaAsO2 treated RegB” reflects
the amount of sulfenic acid in air oxidized RegB⬙, which is ⬃0.4
sulfenic acid per monomer. The disulfide bond content would
likewise be ⬃0.4 per monomer. Finally, the SOH content was
stable 4 °C in air for various periods (1 to 4 day) at ⬃0.4 SOH per
monomer in three independent RegB⬙ preparations. The
remaining ⬃10% of Cys-265 residues unaccounted for likely
represent irreversible oxidation events such as the formation of
SO2H and SO3H.
Sulfenic Acid Formation at Cys-265 in Vivo—We addressed
whether sulfenic acid modification of full-length RegB occurs at
Cys-265 in vivo by exposing cells to DAz-2, which is membrane
permeable (20, 23). DAz-2 was incubated with R. capsulatus
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SB1003 strains carrying chromosomal encoded FLAG-tagged
RegB and RegB C265A mutant that were aerobically cultured.
R. capsulatus cells were then lysed and the FLAG-tagged RegB
and RegBC265A in the cell were enriched using ANTI-FLAG
M2 affinity gel. The enriched FLAG-tagged RegB and RegB
C265A were reacted with p-biotin to conjugate a biotin group
to DAz-2-labeled proteins. Streptavidin-HRP Western blot was
then used to analyze for the presence of biotinylated RegB. The
results in Fig. 5A show that RegB was labeled by DAz-2 in aerobically grown wild type cells but that RegB was not labeled in
the RegB C265A mutant cells. Furthermore, in vivo labeling of
RegB only occurs in cells exposed to air oxygen and not with
cells grown anaerobically (Fig. 5B). These results demonstrate
that Cys-265 is able to form sulfenic acid in vivo in response to
oxygen availability.
Sulfenic Acid Regulates RegB Kinase Activity—We analyzed
the function of a sulfenic acid at Cys-265 by performing an
autophosphorylation assay of isolated RegB⬙ dimer in the presence and absence of DTT. As indicted in the above LC-ESIMS/MS analysis, the RegB⬙ dimer only contains free thiol and
sulfenic acid at Cys-265. Consequently, a change in autophosphorylation observed upon addition of DTT that reduces the
sulfenic acid to free thiol will reflect whether a sulfenic acid at
Cys-265 controls RegB kinase activity. The autophosphorylation assay with isolated RegB⬙ dimer showed that the dimer has
very low autophosphorylation levels in the absence of DTT
while in the presence of DTT the autophosphorylation level is
increased 10-fold in 8 min (Fig. 6). The activation of the RegB⬙
dimer by DTT thus indicates that the presence of a sulfenic acid
at Cys-265 is inhibitory.
DISCUSSION
RegB/RegA system is a major contributor to the metabolic
versatility of R. capsulatus and many other proteobacteria. It
senses the changes in oxygen tension and is responsible for
differential regulation of respiration, fermentation, and photosynthesis gene expression (5, 28). A previous study of the redoxsensing mechanism of RegB disclosed that the fully conserved
cytosolic cysteine, Cys-265 is capable of forming an inhibitory
disulfide bond in response to oxidizing redox potentials (7).
However, it was also shown that only ⬃20% of RegB forms a
tetramer upon exposure to oxygen indicating that other modifications of Cys-265 may occur. Here we present data that a
sulfenic acid also forms at Cys-265 in response to the presence
of atmospheric levels of oxygen in vivo and in vitro. This sulJOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 3. In vitro DAz-2 labeling of RegBⴖ. A, in vitro labeling of wild type
RegB⬙ and RegB⬙ C265A. 2000 ng of wild type RegB⬙ and RegB⬙ C265A were
incubated with 250 ␮M DAz-2 or DMSO at 37 °C for 2 h. 250 ␮M p-biotin then
was added and incubated at 37 °C for 1 h. Labeled samples were resolved by
SDS-PAGE and analyzed by streptavidin-HRP Western blot. The equal loading
was confirmed by anti-STag-HRP Western blot. B, in vitro labeling of reduced
and air oxygen-treated RegB⬙. RegB⬙ was first reduced by 10 mM DTT at 22 °C
for 30 min in anaerobic hood. After removing DTT, 2000 ng of reduced RegB⬙
were exposed to air oxygen at room temperature for 30 min. Treated samples
were labeled and analyzed as in A.
FIGURE 4. Analysis of free and modified thiols in RegBⴖ in vitro. Free thiol
content in air-oxidized RegB⬙, air-oxidized RegB⬙ treated with 10 mM DTT at
22 °C for 1 h, and air-oxidized RegB⬙ treated with 20 mM sodium arsenite at
37 °C for 1 h were determined by chloromercuribenzoate (p-CMB) titration.
Cys-265 Oxidation Inhibits RegB Kinase Activity
FIGURE 6. Effect of the reduction of the sulfenic acid at Cys-265 on RegB
kinase activity. 10 ␮M isolated RegB⬙ dimer was kept at 22 °C for 15 min in the
absence (solid line with open circles) or presence (solid line with solid circles) of
10 mM DTT before [␥-32P]ATP was added to initiate the autophosphorylation
reaction. Aliquots of reactions were removed at 0.5,1, 2, 4, 8 min and
quenched in SDS-PAGE loading buffer, followed by SDS-PAGE separation,
and phosphorimaging data analysis.
fenic acid is not present in cells grown in the absence of air
indicating that it may constitute a reversible redox switch that
directly sensing the oxygen tension. Reduction of the sulfenic
acid to a free thiol clearly increases RegB kinase activity in vitro
indicating that Cys-265 constitutes a SOH/SH redox switch.
Approximately 20% of RegB also forms a disulfide bond leading
to an inactive tetramer in vivo (7). Thus, oxidative modification
of this cysteine is a major contributor to the regulation of RegB
kinase activity.
Sulfenic acids are generally considered highly unstable transient intermediates in catalytic reactions or as intermediates
leading to the formation of more stable oxidation states (29).
However, stable sulfenic acids have been found in proteins that
can be redox regulated (12, 18, 30), suggesting that stability is a
prerequisite for the sulfenic acid to perform the redox-sensing
role. Limited solvent access, active site hydrogen bonding, and
ionization of SOH may be involved in sulfenic acid stabilization;
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FIGURE 5. Formation of sulfenic acid at Cys-265 in vivo. A, formation of
sulfenic acid at Cys-265 in aerobically cultured R. capsulatus. Aerobically cultured R. capsulatus cells carrying RegB-Flag and RegBC265A-flag were incubated with 5 mM DAz-2 or DMSO at 4 °C for 3 h. RegB-Flag and RegBC265Aflag were then enriched with ANTI-FLAG M2 affinity gel, reacted with 250 ␮M
p-biotin, and analyzed by Streptavidin-HRP Western blot. Equal loading was
confirmed by anti-Flag-HRP Western blots. B, formation of sulfenic acid at
Cys-265 in aerobically cultured and anaerobically cultured R. capsulatus cells.
Aerobically cultured and anaerobically cultured R. capsulatus cells were incubated with DAz-2 or DMSO, and the sulfenic acid formation in RegB-Flag was
analyzed as in A.
however the absence of proximal cysteine has been considered
as the dominant factor. The natural tendency of sulfenic acid to
condense with proximal thiols to form disulfide bonds is the
most critical determinant of their fate (18). In the case of RegB⬙,
the sulfenic acid at Cys-265 is highly stable as indicated by the
unchanged sulfenic acid content in different RegB⬙ samples
exposed to air for several days. This stability is manifested by
the non-reducing SDS-PAGE analysis of the isolated RegB⬙
dimer, which showed that the RegB⬙ dimer remains as dimer
even after prolonged exposure to air (Fig. 1B). Cys-265 in solution obviously has the ability to form intermolecular disulfide
bonds, which means Cys-265 from different RegB⬙ molecules
can be placed in proximity; however this ability is abolished in
the RegB⬙ that contains a stable sulfenic acid at Cys-265. Thus,
it appears that once SOH forms at Cys-265, a structural change
must occur to create a protein microenvironment that stabilizes sulfenic acid and/or sequesters it from the Cys-265 from
other RegB⬙ molecules, preventing the formation of disulfide
bonds. Cys-265 is flanked by cationic residues E263, R264,
R266, and D267, which likely reduce its pKa making it highly
redox reactive. Along with Cys-265, these residues are also
highly conserved among RegB homologs indicating that the
oxidation of this Cys is likely a conserved mechanism across a
variety of species (5).
The regulatory roles of cysteine sulfenic acid in enzyme function and redox signaling has only been appreciated recently. Its
formation has been detected in vitro in many proteins, including CrtJ, OxyR, OhrR, the epidermal growth factor receptor
(EGFR), and protein tyrosine phosphatases (11, 13, 14, 16, 18,
31). However capturing the sulfenic acid in vivo has been challenging in most proteins because it is highly unstable and susceptible to further oxidation to more stable states such as disulfide bond, sulfinic acid (SO2H), or sulfonic acid (SO3H). DAz-1
and its enhanced version DAz-2 (20) containing an azide chemical handle, have allowed the detection of sulfenic acid in individual proteins in Rhodobacter capsulatus (31), yeast (32), and
the global analysis of sulfenic acids in tumor cells (23, 33). The
newly described membrane-permeable probe DYn-2 has also
demonstrated the formation of sulfenic acid at a critical Cys
that controls tyrosine kinase activity of EGFR (16). This finding
has implications in tumor biology as some tumor cells are
known to have enhanced protein sulfenylation (33). Controlling membrane spanning kinase activity via sulfenylation now
spans several kingdoms with RegB the first example of a prokaryotic membrane-bound receptor kinase that also has a critical cysteine capable of forming a stable sulfenic acid to control
its activity. Interestingly, RegB is actually a second example of a
regulatory protein in R. capsulatus that utilizes sulfenylation to
control gene expression. In this species, it has recently been
shown that the tetrapyrrole repressor CrtJ stimulates DNA
binding activity in response to sulfenylation of a critical Cys
that is located in the DNA binding domain (31). Thus, the
involvement of sulfenic acid modification of Cys as a means to
control gene expression may be a more common occurrence
than is typically recognized.
Finally, even though this study further demonstrates that
oxidative modification of Cys-265 has a critical role in controlling the activity of RegB, it has also been established that RegB
Cys-265 Oxidation Inhibits RegB Kinase Activity
the redox state of the membrane-located ubiquinone pool
which is linked to oxygen consumption, electron transport, and
energy generation. Structural features of these separate input
signals, such as conservation of Cys-265 and surrounding residues as well as conservation of the ubiquinone binding pocket,
is present in over 200 identifiable homologs of RegB. This indicates that control of this global regulator is highly conserved. It
is likely that the different modes of regulating RegB activity as
shown in Fig. 7 are independent of one another. However, further studies will need to be undertaken to determine is this is
indeed the case.
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FIGURE 7. Model for the regulation of RegB by oxidative modifications of
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