A Low pKaCysteine at the Active Site of Mouse Methionine
Sulfoxide Reductase A*
Jung Chae Lim‡, James M. Gruschus§, Geumsoo Kim‡, Barbara S. Berlett‡, Nico Tjandra§, and Rodney L. Levine‡1
Bethesda, Maryland 20892-8012
Background: The active site cysteine of methionine sulfoxide reductases has been reported to be 9.5.
Results: The pKaof methionine sulfoxide reductase is 7.2.
Conclusion: Methionine sulfoxide reductase has an active cysteine at its catalytic center.
Significance: Methionine sulfoxide reductase is readily oxidized by low concentrations of hydrogen peroxide, supporting both
antioxidant and redox signaling functions of the enzyme.
Methionine sulfoxide reductase A is an essential enzyme in
through cyclic oxidation and reduction of methionine and
methionine sulfoxide. Recently it has also been shown to cata-
lyze the reverse reaction, oxidizing methionine residues to
is essential for both reductase and oxidase activities. This cys-
teine has been reported to have a pKaof 9.5 in the absence of
substrate, decreasing to 5.7 upon binding of substrate. Using
three independent methods, we show that the pKaof the active
site cysteine of mouse methionine sulfoxide reductase is 7.2
even in the absence of substrate. The primary mechanism by
which the pKais lowered is hydrogen bonding of the active site
Cys-72 to protonated Glu-115. The low pKarenders the active
site cysteine susceptible to oxidation to sulfenic acid by micro-
molar concentrations of hydrogen peroxide. This characteristic
supports a role for methionine sulfoxide reductase in redox
A pre ´cis to methionine sulfoxide reductase A (msrA)2is in
the Introduction of the accompanying paper (1). Crystallo-
graphic and solution structures of msrA from various micro-
organisms and the cow established that their active sites
are virtually identical (2–7). These structures and detailed
is always at the active site, and oxidation to the sulfenic acid is
essential for both reductase and oxidase activities (9, 10). The
pKaof the active site cysteine in msrA from Neisseria meningi-
tidis was reported to be ?9.5 in the absence of methionine
sulfoxide, decreasing to 5.7 upon binding of the substrate (11).
A high pKain the absence of methionine sulfoxide could pro-
and nitrogen species (11), but it would also prevent msrA from
(12, 13). In the course of studies on mouse msrA, we observed
sulfenic acid by micromolar concentration of hydrogen perox-
ide. We therefore measured its pKaby three methods, spectro-
photometric titration, oxidation by hydrogen peroxide, and
Preparation of the msrA was described in the accompanying
paper (1). The E115Q mutant of wild-type msrA was con-
structed by recombinant PCR (14). The sulfenamide form of
methionine sulfoxide at room temperature for 5 min (15) fol-
lowed by acidification to pH 1.6 by addition of 1/40 volume of
20% (v/v) trifluoroacetic acid. Acid conditions were chosen to
stabilize the sulfenamide, and cleavage was effected by pepsin
(Sigma P-7012) at a concentration of 4 ?g/?l. The sample was
incubated at 37 °C for 18 h at which time HPLC-tandem mass
spectrometry was performed as described (15) except that the
mass spectrometer was an Agilent model 6200.
Determination of the pKaof Cys-72 by Spectrophotometric
dation of the other three cysteine residues or of Met-229, we
studied the oxidation of Cys-72 with the previously described
Ionization of the cysteine thiol to its thiolate causes an
increase in its molar absorbtivity of ?4,000 M?1cm?1, provid-
ing a direct method for determining pKa(16). Spectra were
recorded on a Shimadzu model 2501 double monochromator
spectrophotometer at 20 °C. Stock msrA was incubated with
dithiothreitol to assure reduction of Cys-72 and then dialyzed
A 100-?l aliquot was mixed with 200 ?l of 60 mM potassium
phosphate, 60 mM sodium pyrophosphate, pH 6.0, to give a
solution of pH 6.2. The msrA concentration was 68 ?M. One
* This work was supported, in whole or in part, by the Intramural Research
Program of the NHLBI, National Institutes of Health.
1To whom correspondence should be addressed: NIH, Bldg. 50, Rm. 2351,
Bethesda, MD 20892-8012. Fax: 301-451-5460; E-mail: firstname.lastname@example.org.
diethylenetriaminepentaacetic acid; HSQC, heteronuclear single-quan-
3The msrA gene encodes both the mitochondrially targeted form and the
cytosolic form. We follow the usual practice in the field and number resi-
cytosolic form whose amino-terminal residue is Gly-22.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 30, pp. 25596–25601, July 20, 2012
Published in the U.S.A.
sample was titrated with 200 mM NaOH, and its pH was mea-
sured after each addition of base. It was important to measure
the pH with msrA present in the buffer because the protein
altered pH by up to 0.2 unit compared with buffer alone. A
second sample was placed in the spectrophotometer, and spec-
tra were measured after each addition of NaOH. The observed
absorbances were corrected for any light scatter and for dilu-
tion by the NaOH. Scatter was calculated from a line fit to the
log of the absorbance measured at 350 and 400 nm (17).
Determination of the pKaof Cys-72 by Its Rate of Oxidation—
Hydrogen peroxide oxidizes the thiolate form of cysteine and
not the thiol form (18). The rate of oxidation of cysteine thus
depends on the hydrogen peroxide concentration, the pH, and
the pKaof the cysteine. As expected, Regino and Richardson
found that the rate of oxidation at pH 8.0 of cysteine, glutathi-
one, and N-acetylcysteine varied in accordance with their pKa
variation (19). However, the rates were the same when calcu-
lated according to the concentration of thiolate present. They
determined the second order rate constant for oxidation of the
thiolate to be 17 M?1s?1at 25 °C. Using the same assay with
cysteine as Regino and Richardson, we found the rate constant
at 37 °C to be 32 M?1s?1, consistent with a simple Q10effect.
order rate of oxidation of msrA, the pKaof the active site cys-
teine can be calculated by measuring the rate of oxidation of
msrA at a single pH from Equation 1 (19),
k ? kobserved
where the rate constant k ? 32 M?1s?1and kobservedis the
observed rate of oxidation by hydrogen peroxide of the active
site Cys-72. This method of determination of pKarequires that
the oxidizing agent employed have unhindered access to the
active site cysteine, a condition that should readily be met by
photometric study was also used for the oxidation studies. The
E115Q mutant of this protein was constructed with a
QuikChange Site-directed Mutagenesis kit (Stratagene) using
the pET17b-C107S/C218S/C227S/?228–233 msrA vector as
GTCGTCCGGGTT-3? and 5?-AACCCGGACGACTTGG-
Oxidations were carried out at 37 °C in 50 mM sodium phos-
phate with 1 mM DTPA. The msrA concentration was 10 ?M,
aliquot of 20 ?l was withdrawn and the oxidation stopped by
immediate mixing with 40 ?l of 50% (v/v) acetic acid.
A convenient method for following the rate of oxidation is
reverse phase chromatography, as the native form elutes later
than the oxidized form. The instrumentation and separation
conditions were as described (10), except that the acetonitrile
gradient was altered to optimize separation. It was brought to
30% (v/v) in 5 min, then to 40% (v/v) over 12.5 min. The native
protein eluted at 14 min with this program. The amount of
native msrA was determined from the area of its peak at 210
Determination of pKaby NMR—Nonmyristoylated13C/15N-
bation with 5 mM dithiothreitol at 37 °C for 30 min. The 300–
400 ?l sample was then dialyzed overnight at 4 °C against two
a pH from 5 to 10.5. The final pH was measured after dialysis.
Constant-time13C heteronuclear single-quantum correlation
(HSQC) was obtained at each pH value.15N HSQC was per-
formed before and after each13C HSQC to confirm sample
proton and carbon resonances, a short time constant (3.5 ms)
for the13C HSQC and a higher temperature (305 K) were used
for pKadetermination, compared with that used for resonance
assignments (300 K) (1). Spectra were processed and analyzed
using nmrPipe (18).
The NMR resonance pH dependence curves were fit using
the Henderson-Hasselbalch equation. For cases where the res-
onance was affected by one ionization, we used
a ? b ? pH ?
?1 ? 10pKa? pH??
For cases where the resonance was affected by two ionizations,
a ? b ? pH ?
?1 ? 10pKa1? pH??
?1 ? 10pKa2? pH?
where a and b are the zero and first order components of the
the chemical shift transitions. Fits were calculated with
Kaleidagraph (Synergy Software).
Determination of the pKaof Cys-72 by Spectrophotometric
Monitoring of Its Ionization—The ionization of Cys-72 was fol-
lowed by the increase in absorbance at 240 nm and gave a typ-
ical Henderson-Hasselbalch plot with pKa? 7.1 (Fig. 1). The
maximal increase in molar absorbtivity from the fit curve was
FIGURE 1. Determination of the pKaof Cys-72 in msrA by absorbance at
240 nm. The symbols (f, F) are the values obtained in two separate experi-
ments on different days. The line is the fit curve for all points, giving a pKaof
JULY20,2012•VOLUME287•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 25597
4,400 M?1cm?1, in good agreement with the value of ?4,000
M?1cm?1estimated by Donovan (16).
Determination of pKaof Cysteines by NMR—The cysteine
pKavalues of msrA were determined from the pH dependence
of their C? resonances, measured by13C constant-time HSQC
(Fig. 2). Fitting to the Henderson-Hasselbalch equation yielded
transitions at 7.2 and 9.7 for Cys-72 and 7.2, 8.4, and 9.6 for
Cys-107, Cys-218, and Cys-227, respectively. The sample pre-
cipitated below pH 6 and was denatured above pH 10.5. The
cysteine backbone amide resonances for15N-labeled myristoy-
lated and nonmyristoylated msrA were also examined. These
exhibited similar chemical shifts and pH dependence (Fig. 3)
from pH 6.5 to 8.5, suggesting that the cysteine pKavalues for
With a limited amount of myristoylated13C/15N-labeled msrA
available, C? resonance pH dependence was measured for the
nonmyristoylated form only.
The pH dependence for C? and the backbone amide of
Cys-72 both show two transitions, one at pH 7.2, and a larger
one at 9.7 (Figs. 2 and 3). Such pH profiles are expected if, in
addition to ionization of its thiol, a cysteine residue interacts
with another titratable group such as a tyrosine, aspartate, or
one of the two transitions, but the spectrophotometric method
and the oxidation method do allow us to assign the first transi-
tion to Cys-72 ionization.
We note that Cys-72 lies near the carboxylate group of Glu-
115. This carboxylate group is hydrogen-bonded to the back-
bone amide of the following residue, Phe-73. Examination of
115 shows a clear transition at pH ?7.2 for both (Fig. 4). The
carboxylate of Glu-115 is ?5 Å from the SH group of Cys-72,
cause the pH 7.2 feature in the Cys-72 pH-dependence curve
(21). This possibility was investigated experimentally (see later
subsection). The thiol of Cys-107 also lies near Glu-115, ?8 Å
influenced by Glu-115. The curve for Cys-107 also shows an
The backbone amide signal of Trp-74 shows a strong transi-
tion at pH 9.7, with perhaps a weaker transition at approxi-
mately pH 7.2 (Fig. 4). A rotation of the Cys-72 side by ?60
degrees brings the Cys-72 sulfur within hydrogen-bonding dis-
ing that Cys-72 in the thiolate form can facilitate a hydrogen
of Cys-72 to form a sulfenamide (10). The nitrogen participat-
ing in sulfenamide formation is probably a peptide bond amide
because there are no amines near Cys-72. The Cys-72 sulfur is
FIGURE 2. pH profiles of the13C chemical shifts of the C? atoms of cys-
as the residue name.
FIGURE 3. pH profiles of the1H chemical shifts of the backbone amide
atoms of cysteines in msrA. Filled symbols, myristoylated msrA; open sym-
bols, nonmyristoylated msrA.
FIGURE 4. pH profiles of the1H chemical shifts of the backbone amide
atoms of residues close to the active site Cys-72.
adjacent Phe-73 and Trp-74, making them the most likely can-
didates to form the sulfenamide.
We determined the site of sulfenamide formation by peptic
mapping. In the nonoxidized control msrA, pepsin cleavage
occurred after Phe-68 and Phe-73, placing Cys-72 in the
peptide G69M70G71C72F73 (calculated mass ? 513.1716,
observed ? 513.1731). As expected, this peptide was not
observed in the sulfenamide form of msrA. Trp-74 in the control
a peptide of mass 511.1716 should be observed. If Trp-74 formed
the sulfenamide, then the two linked peptides would have mass
tide, but it did have a new peak at 972.3471, establishing that the
sulfenamide was formed between Cys-72 and Trp-74. This was
confirmed through sequencing by tandem mass spectrometry
(data not shown). We conclude that the thiolate of Cys-72 is sta-
The pH-dependent shifts for Gly-71, Gly-75, and Ala-76, as
well as the side chain amide of Trp-74 show relatively small
changes (?0.2 ppm) and no significant broadening, suggesting
occurring in the pH range studied.
Determination of the pKaof Cys-72 by Its Rate of Oxidation—
Cysteine pKavalues in proteins have often been estimated by
for the thiolate form such as alkylation by iodoacetamide. The
rates be determined and that the reagent have unhindered
access to the cysteine residue of interest, conditions that are
often not met. Hydrogen peroxide generally has access to all
solvent-exposed residues, and Cys-72 is such a residue (2–7).
We measured the rate of oxidation of Cys-72 by hydrogen per-
rate was linear when displayed on a semilogarithmic plot (Fig.
5). As noted under “Experimental Procedures,” this method
allows determination of the pKafrom the oxidation rate at a
was measured at varying pH with the same result (Fig. 5, inset).
The pKaof Cys-72 was 7.4, in good agreement with 7.1 and 7.2
measured by spectrophotometric and NMR titration. The
agreement among the three methods supports the conclusion
of the three methods.
3) and could also lower the pKaof Cys-72 if Glu-115 were pro-
tonated and formed a hydrogen bond with Cys-72 (22, 23).
Because we observed that Glu-115 and Cys-72 share a transi-
tion with pKaof 7.2, we studied the susceptibility to hydrogen
peroxide of the E115Q mutant because the hydrogen bond
ing that Glu-115 lowers the pKaof Cys-72.
Our assays of activity of msrA are routinely performed at pH
7.5. When the E115Q was assayed at this pH, its activity was
only ?2% that of the Glu-115 wild-type form. If the reduced
activity were due simply to the higher pKaof Cys-72, then the
ity would be lowered across the pH activity curve. Fig. 7 shows
that the pH activity profiles of the wild-type and mutant are
superimposable, but the mutant has only ?2% of the activity of
the wild-type at all pH values. We conclude that Glu-115 is
essential both for lowering the pKaof Cys-72 and for substrate
binding, as suggested by Ref. 11.
Biochemical, mutational, and structural studies have firmly
of Cys-72 at physiological pH requires that the typical cysteine
pKaof 8.5–8.8 be lowered. “Active” or low pKacysteines at the
FIGURE 5. Determination of the pKaof Cys-72 in msrA by the rate of its
oxidation by hydrogen peroxide. The pseudo-first order rate constant for
the oxidation at pH 7.01 is shown in the semilogarithmic plot. The observed
second order rate constant and the calculated pKaat different pH values are
given in the table inset.
its oxidation by hydrogen peroxide. The pseudo-first order rate constant
for the oxidation at pH 8.21 is shown in the semilogarithmic plot. The
observed second order rate constant and the calculated pKaat different pH
value are given in the table inset. The msrA used in this study was
JULY20,2012•VOLUME287•NUMBER30 JOURNALOFBIOLOGICALCHEMISTRY 25599
the pKais often effected by nearby basic residues which facili-
tate deprotonation. None of the structures of msrA from a
number of species has this feature at their active site. Cys-72 is
located at the amino-terminal end of an ?-helix. The two
groups reporting the first structures of msrA, one from Esche-
richia coli (3) and the other from the cow (2), both suggested
that this placement could decrease the pKaof msrA. The low-
ering of the pKaoccurs because the amino-terminal side is the
positive end of the ?-helix dipole (24, 25). A protonated car-
thiolate through hydrogen bonding. This less common struc-
tural mechanism for lowering the pKais well documented to
functions to lower the pKafor protein splicing by inteins (22).
strates that hydrogen bonding of protonated Glu-115 is the
major mechanism by which the pKaof Cys-72 is lowered.
observed facile oxidation of the active site Cys-72 by hydrogen
peroxide at neutral pH. This observation implied that its pKa
was low even in the absence of substrate. With the recognition
that murine msrA is also a stereospecific protein methionine
oxidase (10), a reinvestigation of the pKaof Cys-72 became
important. If hydrogen peroxide in the absence of substrate
could react with msrA to form the sulfenic acid, then the en-
zyme could participate in redox signaling. We therefore mea-
sured the pKaby spectrophotometric titration, by NMR titra-
tion, and by the rate of oxidation by hydrogen peroxide. Each
method demonstrated a low pKa, with an average of 7.2. NMR
indicated that Cys-72 and Glu-115 appear to be coupled titrat-
able groups, thus the first transition at 7.2 corresponds to loss of
the first proton of this two-proton system. Additional investiga-
tions will be required to elucidate the structural basis for the sec-
The apparent pKaof msrA from N. meningitidis has been
studied by two methods (11). The first was the rate of reaction
of the active site cysteine with dipyridyl disulfide which gave a
reagent to the active site cysteine might yield an inaccurate
pendent change in absorbance at 240 nm of msrA and again
obtained a high pKaof 9.7. The increase in molar absorbtivity
Donovan first reported the spectrophotometric titration at
240 nm of cysteines in aldolase (16). He found that the molar
absorbtivity increased by ?4,000 M?1cm?1upon ionization.
However, he also pointed out that tyrosine residues also show
an increase in molar absorbtivity at 240 nm upon ionization,
and that increase was approximately 11,000 M?1cm?1. The N.
tyrosine residues. Thereportedincreaseinabsorbtivityof23,000
ization. The observed pKaof 9.7 is typical for tyrosine residues.
topyridine suggests that tyrosine ionization was required for
The first crystal structures of msrA led to the proposal that
Glu-115 was important in binding and orientation of the sulf-
oxide substrate (2, 3). Subsequent investigations by Branlant
and colleagues provided evidence that Glu-115 was also
involved in the activation of Cys-72, but only after binding of
substrate is not required to lower the pKaof Cys-72.
Signaling and regulation by redox modification of proteins
are now well established physiological mechanisms (12, 13).
Recently, msrA was found to catalyze the oxidation of protein
alyzing reduction of the sulfoxide, and a structural basis for
regulation of this bifunctional enzyme was proposed (10). The
oxidizing agent was millimolar methionine sulfoxide, a non-
physiological concentration. Our present study demonstrates
that the active site cysteine of msrA can be converted to its
peroxide. Thus, msrA may participate in a regulatory system
that is dependent on hydrogen peroxide.
Considerable experimental evidence supports a role for
msrA in oxidative defense by scavenging reactive oxygen and
nitrogen species through cyclic oxidation and reduction of
methionine and methionine sulfoxide (26–35). The presumed
pathway of scavenging is via direct oxidation of protein methi-
onine residues by the reactive species, following which msrA
hypochlorous acid reacts well with methionine at physiological
pH (36, 37), hydrogen peroxide does not, although the rate can
be accelerated by the bicarbonate/carbon dioxide present in
vivo (38). Acting in the oxidase direction, msrA can react with
low concentrations of hydrogen peroxide to form sulfenic acid
dize methionine residues in proteins (10). Subsequently, acting
in the reductase direction, msrA can utilize thioredoxin to
reduce the protein methionine sulfoxides back to methionine.
The net result is that the low pKaallows msrA to scavenge
hydrogen peroxide, thus contributing to oxidative defense.
FIGURE 7. pH profile for reduction of methionine sulfoxide by wild-type
(f) and E115Q (F) msrA. The wild-type activity is plotted against the left
ordinate and the E115Q activity against the right ordinate.
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