Available online at www.sciencedirect.com
Expanding the functional diversity of proteins through cysteine
oxidation
Khalilah G Reddie1 and Kate S Carroll1,2
The polarizable sulfur atom in cysteine is subject to numerous
post-translational oxidative modifications in the cellular milieu,
which regulates a wide variety of biological phenomena such as
catalysis, metal binding, protein turnover, and signal
transduction. The application of chemical rationale to describe
the features of different cysteine oxoforms affords a unique
perspective on this rapidly expanding field. Moreover, a
chemical framework broadens our understanding of the
functional roles that specific cysteine oxidation states can play
and facilitates the development of mechanistic proposals,
which can be tested in both biochemical and cellular studies.
Addresses
1
Life Sciences Institute, University of Michigan, Ann Arbor, MI 481092216, United States
2
Department of Chemistry, University of Michigan, Ann Arbor, MI 481092216, United States
Corresponding author: Carroll, Kate S (katesc@umich.edu)
Current Opinion in Chemical Biology 2008, 12:746–754
This review comes from a themed issue on
Model Systems
Edited by Helma Wennemers and Ronald T. Raines
Available online 17th September 2008
1367-5931/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2008.07.028
Introduction
Reduction and oxidation comprise an important class of
post-translational modifications. Reactive oxygen species
produced from a variety of sources, such as the mitochondria and NADPH oxidases, modulate the activity of
proteins. In this context, the thiol side chain of cysteine
is most sensitive to redox transformations and can occur in
a variety of oxidation states. Among these, the thiol and
the disulfide are best known, but oxygen derivatives such
as sulfenic (RSOH), sulfinic (RSO2H), and sulfonic
(RSO3H) acid are observed in a growing number of
proteins. In this review, we discuss the chemical properties of these cysteine oxoforms and summarize recent
studies that highlight ways in which these modifications
are exploited in the cellular milieu to increase functional
diversity in proteins.
The chemistry of cysteine
The amino acid cysteine is distinguished by a thiol
functional group, which contains a sulfur atom and a
Current Opinion in Chemical Biology 2008, 12:746–754
hydrogen atom (RSH). The large, polarizable sulfur atom
in a thiol group is electron-rich and quite nucleophilic. Its
reactivity is enhanced in the deprotonated form of the
thiol, also known as the thiolate anion (RS). In this
regard, the thiol group is mildly acidic and the pKa value
is dependent on the structure and local environment. In
peptides the pKa is typically 9, but in proteins this value
can be as low as 3.5 [1]. The thiolate anion reacts with
‘hard’ electrophilic centers, such as the carbonyl, phosphoryl, and sulfuryl (O2SX2) groups, as well as with ‘soft’
electrophiles such as saturated carbon. Owing to its high
reactivity, the thiol group of cysteine plays many important biological roles in catalysis, metal binding (e.g. Zn2+
and Fe2+), and detoxification of xenobiotics. In addition,
the thiol side chain of cysteine is widely used as a
nucleophile for many post-translational modifications including S-acylation and protein splicing.
Cysteine oxidation in proteins
Sulfur is the second-row element of group VIa of the
periodic table with an electron configuration of
[Ne]3s23p4. The availability of d-orbitals for bonding in
sulfur allows expansion beyond divalent compounds to
valencies of 4 and 6 at oxidation states ranging from 2 to
6+. Some of these species, which can occur under physiological conditions, are shown in Figure 1. In cysteine,
the sulfur atom is fully reduced with an oxidation state of
2. Owing to its low oxidation–reduction (redox) potential in proteins (E80 0.27 to 0.125 V) the thiol side
chain is readily oxidized, which plays a significant role in
its chemical and biological function [1].
The most commonly known thiol oxidation reaction is
disulfide formation (Eqn (1)):
(1)
2RSH ! RSSR þ 2e þ 2Hþ
Disulfide bonds are reasonably stable intermediates and
are often involved in the maintenance of protein structure
and function. For example, interchain disulfide bridges
between heavy and light chains stabilize the immunoglobin protein fold in antibodies [2]. Compared to thiols,
the chemistry of RS–SR bonds are fairly limited. However, disulfides will undergo nucleophilic cleavage and
thiol–disulfide exchange (Eqn (2)), which is an extremely
important biological reaction [1]:
(2)
RSSR þ R0 S ! RSSR0 þ RS
Other oxidation states of cysteine between 1 and 4+ are
energetically less stable and the free energy for further
oxidation is high. In addition, as oxidation numbers
increase, the sulfur atom in cysteine acquires a greater
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Expanding the functional diversity of proteins through cysteine oxidation Reddie and Carroll 747
Figure 1
been identified in a growing list of proteins and they have
received intense interest for their roles in enzyme catalysis and cell signaling ([4] and references therein).
Sulfenic acids are formed by the reaction of a thiol or
thiolate anion with hydrogen peroxide (Eqn (3)) and other
biological oxidants such as peroxynitrite. Sulfenic acids
can also result from the hydrolysis of S-nitrosothiols (Eqn
(4)), after the reaction of thiol or thiolate anions with
thiolsulfinates (Eqn (5)), and the condensation of a thiyl
and hydroxyl radical (Eqn (6)). Finally, sulfenic acids are
generated via reaction of a thiol or thiolate anion with
hypochlorus acid ([5] and references therein) (Eqn (7)),
which is enzymatically produced in human immune cells
during the inflammatory response. In kinetic studies, the
pKa for the sulfenyl group of cysteine sulfenic acid has
been estimated to be between 6 and 10 [5]:
RSH þ H2 O2 ! RSOH þ H2 O
RSNO þ H2 O ! RSOH þ HNO
(3)
(4)
RSH þ RSðOÞSR ! RSOH þ RSSR
(5)
Oxidative post-translational modifications of cysteine. The oxidation
number of the sulfur atom in each of the cysteine oxoforms is shown.
positive charge (Figure 2) and becomes less nucleophilic
in character. The variety of oxidation states, each with
different chemical reactivity and metal-binding properties, combine to make the chemistry and biology of
cysteine oxidation a fertile area of research.
Sulfenic acids
Among the different modifications generated by thiol
oxidation, sulfenic acids (RSOH) are implicated in a wide
variety of important chemical [3] and biochemical reactions [4]. Sulfenic acids are unstable and highly reactive
functional groups, which have traditionally been viewed
as intermediates en route to other oxidation states
(Figure 1). In recent years, however, sulfenic acids have
RS þ HO ! RSOH
(6)
RSH þ HOCl ! RSOH þ HCl
(7)
Reported oxidation rates for sulfenic acid formation in
proteins via reaction with hydrogen peroxide (the most
abundant and stable reactive oxygen species in the cell)
range from 10 to 107 M1 s1 ([6] and references therein).
The reactivity of the thiol side chain is a function of its
ionization state and thiolate anion stability, which is
modulated by the surrounding protein microenvironment. Many redox-sensitive proteins, such as glyceraldehyde-3-phosphate dehydrogenase, tyrosine phosphatases,
and proteases contain active-site cysteine residues that
are essential for function and thus, are inhibited by the
oxidation of these key cysteine thiols ([6] and references
therein). In some cases, oxidation can play an activating
role, as in Hsp33, which acquires chaperone function after
the release of a zinc metal ion [7]. As we will see in the
sections below, oxidative activation and inactivation can
Figure 2
Electrostatic potential surface of sulfur in oxidation states 2, 0, 2+ and 4+ representing a thiolate, sulfenate, sulfinate, and sulfonate, respectively (a–
d). The surfaces depict the highly delocalized negative charge of a nucleophilic thiolate, and the increased positive charge on sulfur with increased
oxidation state. Electrostatic potential surfaces were generated using Spartan 06 (Wavefunction, Inc.). Color gradient: red corresponds to most
negative and blue corresponds to most positive.
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Current Opinion in Chemical Biology 2008, 12:746–754
748 Model Systems
Scheme 1
be reversible or irreversible, depending on the cysteine
oxidation state that is formed.
In early reports, limited solvent exposure was thought to
be required for protein sulfenic acid formation [8]. However, this long-standing rule has recently been revisited.
Salsbury et al. reported the first study to apply functional
site profiling and electrostatic analysis to identify features
that affect the likelihood of protein cysteines to form
sulfenic acids [9]. By analyzing the sequence and structure at known sites of sulfenic acid formation in proteins,
the authors discovered that solvent inaccessibility does
not correlate with the propensity for cysteine oxidation.
Rather, Salsbury et al. found that polar residues, but not
necessarily charged residues, as well as the presence of
histidine and threonine were important factors for
cysteine oxidation [9].
Sulfenic acids exhibit both electrophilic and nucleophilic
chemical reactivity. This dual nature is highlighted by
thiosulfinate (RS(O)SR) formation, which proceeds
through a hydrogen-bonded sulfenic acid dimer and is
the most common reaction between unhindered sulfenic
acids (Scheme 1). Sulfenic acids react with carbon nucleophiles such as alkenes and enolates including the reagent
5,5-dimethyl-1,3-cyclohexadione, also known as dimedone. The mechanism of this reaction has not been
studied in detail, but could proceed via 1,4-addition or
direct nucleophilic substitution (Scheme 2). This chemical reaction has recently been exploited for the design of
biotinylated and fluorescent reagents for sulfenic acid
detection ([4] and references therein) and for a cellpermeable probe that enables trapping and tagging sulfenic acid-modified proteins directly in living cells [10].
The electrophilic sulfur atom in sulfenic acid leads to one
of its most important biological reactions, which is its
condensation with a nearby cysteine thiol on a protein
(Eqn (8)) or with a low-molecular-weight thiol such as
GSH (Eqn (9)) to form a disulfide:
RSOH þ R0 SH ! RSSR0 þ H2 O
RSOH þ GSH ! RSSG þ H2 O
(8)
(9)
In model chemical studies, the lower limit for the reaction
of unhindered cysteine and cysteine sulfenic acid was
recently estimated at 105 M1 s1 [5]. Cysteine oxidation, leading to the formation of covalent disulfide
bridges, can modulate protein structure and function.
Moreover, disulfides are reversible through the action
of cellular reductants such as glutathione (GSH) and
thioredoxin (Trx). Intramolecular and intermolecular disulfide formation vis-à-vis a sulfenic acid intermediate
plays an essential activating and/or protective role in a
Scheme 2
Scheme 3
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Expanding the functional diversity of proteins through cysteine oxidation Reddie and Carroll 749
wide variety of proteins including OxyR [11], Yap1 [12],
NF-kB [13], and AP-1 [14] transcription factors, Hsp33
chaperone [7], SENP1 SUMO protease [15], E2
SUMO ligase [16], as well as components of the cytoskeleton [17].
When fixed in close proximity, the sulfur atom in a
sulfenic acid can also react with an amide nitrogen to
form a cyclic sulfenamide (Scheme 3). This unusual
modification was first directly observed in a crystal structure of protein tyrosine phosphatase PTP1B [18] and
more recently in RPTPa [19]. Generation of the cyclic
sulfenamide inhibits PTP catalytic activity by blocking
the nucleophilic cysteine residue and is accompanied by
significant changes in the catalytic site and pTyr loops.
Gates and coworkers developed a benzanilide-derived
sulfenic acid model system, which showed that the sulfenic acid is sufficiently electrophilic to drive the cyclization reaction [20]. Sarma and Mugesh have recently
investigated the role of steric and electronic environments around the sulfur and nitrogen atoms and of nonbonded S O/N interactions on the cyclization
reaction in substituted benzene sulfenic acids [21]. Sulfenamide oxidation products of sulfenic acid are reversible and, like disulfides, these modifications may serve a
protective role by preventing overoxidation of cysteine
residues to sulfinic or sulfonic acid.
One of the most important factors in stabilizing a sulfenic
moiety is the absence of proximal thiol groups or other
nucleophiles. If reduction is prohibited, sulfenic acids can
persist in proteins for several hours, as in human serum
albumin Cys34 [22]. On the basis of crystallographic
studies Nakamura et al. have recently reported an intriguing new proposal for sulfenic acid stabilization in an
archaeal 2-Cys peroxiredoxin (Prx) [23]. In their structure, the sulfenic acid form of the Prx is hypervalent, with
the Sg of the cysteine sulfenic acid intermediate 2.2 Å
away from the nitrogend1 atom of a neighboring histidine
residue (Figure 3). The authors propose that the unique
Sg–Nd1 covalent bond prevents overoxidation of the
cysteine sulfenic acid intermediate during the catalytic
cycle [23]. Key features of this hypothesis remain to be
tested in functional studies. In particular, it is not clear
Figure 3
Hypervalent sulfur intermediate formed between the Sg atom of Cys50
and the Nd1 of His42 is central to the stabilization and prevention of
overoxidation of the sulfenic acid in Aeropyrum pernix K1 (ApTPx). Key
structural features include the angle defined by O, S, and N (170 4.58)
and the angle defined by O, S, and C nearly perpendicular at 81.3 2.88.
Dissociation of the hypervalent intermediate leads to the formation of a
cysteine sulfenic acid. Both the hypervalent sulfur intermediate and the
resulting sulfenic acid contain S in oxidation state 0. Figure generated in
Pymol using PDB code 2ZCT and related structure factors for ApTPx.
whether the hypervalent intermediate is susceptible to
reduction by the resolving cysteine or if the sulfenic acid
must be formed before the disulfide (Scheme 4). Furthermore, the active-site histidine residue is not universally
conserved and, thus, it is not known how general the
proposed hypervalent stabilization mechanism will be.
Collectively, the sulfenic acid modification has now been
observed in more than 30 protein crystal structures ([9]
and references therein) and, for the first time, in a
synthetic peptide comprised of the matrix metalloproteinases (MMP-7) cysteine switch domain using tandem
mass spectrometry [24]. At the present time, there is
no direct estimate for the levels of sulfenic acid modifiedproteins in resting or stimulated cells and, of these,
the subset of proteins for which this modification has a
Scheme 4
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Current Opinion in Chemical Biology 2008, 12:746–754
750 Model Systems
Scheme 5
functional role. New ratiometric methods for the analysis
of the sulfenic acid proteome, in conjunction with biochemical analysis, will be required to address these questions.
Sulfinic and sulfonic acids
In addition to their role as key intermediates in the
formation of disulfides, sulfenic acids are intermediates
in the oxidation of thiols to sulfinic (RSO2H) and sulfonic
(RSO3H) acids (Eqn (10)):
½O
½O
RSOH!RSO2 H!RSO3 H
(10)
In fact, it has been estimated that 5% of cellular protein
cysteines occur in the sulfinic or sulfonic acid form [25].
Sulfinic acid can be formed from sulfenic acid by reacting
with oxidants, such as hydrogen peroxide or via thiosulfinate disproportionation [26]. With a pKa 2, cysteine
sulfinic acid is deprotonated at physiological pH and
the sulfinate anion can be represented in two ways
(Scheme 5). DFT calculations using ethane sulfinate as
a model system suggest that both resonance forms contribute equally; the minimized structures show no difference in energies, bond length or charge distribution on
the sulfinate (KS Carroll, unpublished data).
Unlike sulfenic acids, sulfinic derivatives do not undergo
self-condensation reactions or react with thiols under
physiological conditions. At first glance, the lack of reactivity may seem counterintuitive. However, the increase
in partial positive charge on sulfur (Figure 2) converts the
sulfur atom in sulfinic acid into a harder electrophile,
which is less prone to react with soft nucleophiles.
Remarkably, sulfinates (RSO2) still behave like soft
nucleophiles and will undergo alkylation, as well as
nucleophilic addition to activated alkenes, aldehydes,
lactones, and a,b-unsaturated compounds to form the
corresponding sulfones (Scheme 6) [26]. If activated by
an adjacent keto functional group, sulfinic acids will
hydrolyze to sulfite (SO32). For example, the spontaneous hydrolysis of b-sulfinyl pyruvate is a key step
in cysteine catabolism [27]. Other naturally occurring lowmolecular-weight sulfinic acids include cysteine sulfinate
and hypotaurine.
In proteins, the sulfinic acid modification has received the
most attention in the family of Prxs [28,29], which reduce
hydrogen peroxide and alkylperoxides to water and alcohol. Functional roles for sulfinic acid modifications have
also been identified in numerous proteins including in
D-amino acid oxidase (DAO) [30], Parkinson’s disease
protein DJ-1 [31], and the copper–zinc superoxide dismutase (SOD1) [32].
Cysteine oxidation to sulfinic acid can modulate protein
metal binding properties. In matrix metalloproteases
(MMPs), oxidation of an active-site zinc thiolate ligand
to sulfinic acid activates protease function [33]. Another
interesting example of a sulfinic acid that modulates
protein metal binding activity has been identified in
the nonheme iron enzyme nitrile hydratase (NHase),
which hydrolyzes nitriles to amides. In this protein,
two cysteine residues coordinated to the metal are modified to sulfinic and sulfenic acids; biochemical studies
indicate that oxidation of these ligands is essential for
catalytic activity (Figure 4) [34,35].
To probe the functional consequences of cysteine ligand
oxidation in catalysis by NHase, Solomon and colleagues
Scheme 6
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Expanding the functional diversity of proteins through cysteine oxidation Reddie and Carroll 751
Figure 4
oxidation examples of this phenomenon will probably
increase over time.
Unlike thiols and sulfenic acids, sulfinic acids cannot be
reduced by major cellular reductants such as GSH and
Trx. In fact, sulfinylation of a protein cysteine was considered to be irreversible until the discovery of a new
ATP-dependent enzymatic sulfinic acid reductase,
referred to as sulfiredoxin (Srx) [37]. In terms of substrate
specificity, Srx appears to be a dedicated enzyme for the
reduction of sulfinic acid in the Prx family. Lowther and
colleagues have reported the crystal structure of the
human Srx–PrxI complex and reveals how local unfolding
of the Prx active exposes the sulfinic acid for reduction
[38]. Accompanying biophysical studies suggest that the
large-scale conformational rearrangement in Prx is
mediated by Srx binding [38].
Nonheme iron active-site center of Rhodococcus sp. N-771 showing
coordination to NO, three cysteine residues (109, 112, and 114) and also
the main chain amide nitrogens of Cys114 and Ser113. Two cysteine
residues, Cys114 and Cys112, are post-translationally modified to
sulfenic and sulfinic acid, respectively. The metal center is adjacent to
residues Arg56 and Arg141, which hydrogen bond to oxygens of the
modified cysteines. Blue, brown, yellow, gray, and red spheres
represent nitrogen, iron, sulfur, carbon, and oxygen, respectively. Figure
generated in Pymol using PDB code 2AHJ.
recently investigated the geometric and electronic structure of the active site via sulfur K-edge XAS and DFT
calculations on synthetic model complexes [36]. These
studies demonstrate that the edge region of the spectra
shows dramatic differences upon oxidation and, in the
case of sulfenate, the edge is also sensitive to protonation
state. Since oxidized sulfur ligands are weaker donors that
can increase the Lewis acidity of the FeIII center, the
authors of this study propose that cysteine oxidation
modulates the binding affinity of the catalytically relevant
water molecule to a vacant coordination site [36]. Few
examples of protein active sites with transition metals
coordinated to oxidized cysteine residues have been
reported, however, with the growing interest in cysteine
Two mechanisms have been proposed for Srx-catalyzed
sulfinic acid reduction [37,39] and these differ in the first
step of the reaction. In the original proposal by Biteau
et al., the g-phosphate of ATP is directly transferred to Prx
(Scheme 7) [37]. Alternatively, Jeong et al. suggested that
Srx served as a phosphorylated intermediary, en route to
Prx phosphorylation [39]. Although details regarding the
role of Srx differ in each mechanism, both mechanisms
involve the formation of unique phosphoryl sulfinic
enzyme intermediate in Prx (Scheme 7). Literature precedent shows that sulfinic acid oxygens can indeed act as
nucleophiles in phosphoryl ester formation [40]. The
most recent mechanistic [41] and structural [42] studies
support the original mechanistic proposal [37], where the
phosphorylation of Prx sulfinic acid is the first chemical
step. Several issues remain unresolved in this interesting
reaction, including the nature of chemical steps beyond
sulfinyl phosphoryl intermediate formation and whether
any accessory proteins enhance the sluggish rate of
reduction observed with Srx alone in vitro, currently
estimated at 0.2 min1 [43].
Sulfinic acids are stable intermediates, but oxidize readily
to sulfonic acid (RSO3H), the most highly oxidized
species of thiols and disulfides (Eqn (10) and
Figure 1). Strong oxidizing agents such as halogens,
hydrogen peroxide, and nitric acid can generate sulfonic
acids from thiols [26]. In addition to proteins, sulfonic
Scheme 7
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Current Opinion in Chemical Biology 2008, 12:746–754
752 Model Systems
Scheme 8
acids are found in many naturally occurring, low-molecular-weight compounds such as taurine, isethionic acid,
and methansulfonic acid [27]. Owing to the stability of the
conjugate base, which can stabilize the negative charge
through resonance localization, sulfonic moieties are
strong acids comparable to sulfuric acid in strength
[26]. As weak bases, sulfonates (RSO2O) are good leaving groups in SN1, SN2, E1, and E2 reactions. In organic
synthesis, the sulfonic acid group is often used as a
directing group or is installed on hydrophobic molecules
as a method to improve solubility of a compound.
Irreversible oxidation of N-terminal cysteine to sulfinic or
sulfonic acid can impact protein function and homeostasis
in many ways. For example, the introduction of highly
oxidized sulfur species with distinct negative charge
distribution and steric requirements can result in changes
to protein structure. Sulfinic and sulfonic acid modifications can inhibit the activity of enzymes that require a
thiolate for catalysis. Alternatively, the oxidation of a
cysteine thiol to a sulfenic, sulfinic acid, or sulfonic acid
can also be a prerequisite for proper protein function, as
highlighted by several examples above. Irreversible
cysteine oxidation can also target a protein for degradation, as in the N-end rule pathway. In this system, the
oxidation of N-terminal cysteine residues to sulfinic and
sulfonic acid in certain mammalian proteins, such as
GTPase-activating proteins (RGS), is required for arginylation by ATE1 R-transferases and subsequent ubiquitin-dependent degradation (Scheme 8) ([44] and
references therein).
Conclusions
The expanding repertoire of proteins that contain redoxbased cysteine post-translational modifications highlights
their growing functional significance. Each cysteine-containing protein whose function is modified in response to
ROS in vitro now becomes a candidate for a redoxregulation. Chemical approaches exist to identify proteins
modified to disulfides ([45] and references therein) and,
more recently, sulfenic acids ([4] and references therein,
[10]). Recent improvements in the detection of higher
cysteine oxidation states by tandem MS–MS [46] and
electron capture dissociation MS [47], as well as antibodies generated against peptides containing cysteine
sulfinic/sulfonic acids [48–50] should facilitate the investigation of the biological roles of higher oxidation states.
The continued development of methods that enable in
Current Opinion in Chemical Biology 2008, 12:746–754
situ analysis of individual cysteine oxoforms will be
essential to investigate the role of these modifications
in physiologically relevant settings.
Acknowledgements
We apologize to those authors whose work we could not cite due to space
limitations. We thank the Life Sciences Institute, the Leukemia &
Lymphoma Society Special Fellows Award #3100-07 and the American
Heart Association Scientist Development Grant #0835419N to KSC for the
support of this work. We also thank Candice Paulsen and Donald Raymond
for their assistance with figures.
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754 Model Systems
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