Protein S-Thiolation by Glutathionylspermidine (Gsp)

Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated by H2O2 oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA and GspSA/glutaredoxin null mutants to H2O2 support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli.

Protein thiols are readily oxidized and reduced to form sulfenates, sulfinates, sulfonates, and intra-and intermolecular disulfides. Most organisms have complex systems that regulate the intracellular redox states of thiols (1,2). Small thiol-containing biomolecules (e.g. GSH and cysteine, form mixed-disulfides with protein thiols (S-thiolation). These post-translational modifications protect proteins from overoxidation and regulate certain protein functions (3,4). For example, S-glutathionylation of Escherichia coli methionine synthase, which occurs when E. coli is oxidatively stressed, suppresses the synthase activity, thereby decreasing cellular methionine concentration (5). Because GSH is abundant in most organisms (often existing at 1-10 mM), protein S-glutathionylation (GSH S-thiolation) is considered to be a reversible and universal cellular process. In E. coli, GSH and spermidine (Spd) 4 form N 1 -glutathionylspermidine (Gsp) via an ATP-dependent reaction catalyzed by the C-terminal Gsp synthetase domain (supplemental Fig. S1) (6 -8). E. coli GspSA also hydrolyzes Gsp to GSH and Spd via the N-terminal amidase domain (supplemental Fig. S1) (6). Although GspSA was first found in E. coli more than 3 decades ago (9), it is not known what the physiological role of Gsp is or how the two opposing activities of GspSA are regulated in vivo.
Herein, we report that Gsp S-thiolated proteins (GspSSPs) have mixed disulfides of Gsp and protein thiols in E. coli. Intriguingly, we found that the level of GspSSPs increased when E. coli was treated with H 2 O 2 , indicating that this modification probably inhibits oxidation of protein thiols. The accumulation of GspSSPs probably occurred because, although Gsp amidase was inactivated by H 2 O 2 , Gsp synthetase was mostly unaffected. According to an x-ray crystallography study and a chemical modification study, we found that H 2 O 2 oxidized the thiol of the amidase active-site nucleophile, Cys 59 , to a sulfenic acid and that the sulfenic acid was stabilized by several hydrogen bonds, one of which involved a water molecule and was unusually short. We propose a set of reactions that explain how the transient inactivation of Gsp amidase leads to an accumulation of Gsp and an increased level of GspSSPs after oxidative stress. With elimination of the oxidative threat, Gsp amidase, GSH reductase, and glutaredoxin act in concert to convert oxidized Gsp (as the disulfide of Gsp ((GspS) 2 ), mixed disulfides of Gsp, and other small thiol-containing compounds and/or GspSSPs) to GSH. That GspSA and glutaredoxin act synergistically is supported by the hypersensitivity of E. coli mutants that lack both enzymes to H 2 O 2 .

EXPERIMENTAL PROCEDURES
The following experiments are described in the supplemental material due to space limitations: the construction of GspSA disrupted strain, protein crystallization and refinement, identification of sulfenic acid by dimedone, and the hydrolysis of GspSSPs by Gsp amidase.
Detection of E. coli Gsp S-Thiolated Proteins-NR754 (wild type) and HA61002 (⌬gspSA) were each cultured in 1 ml of M9 minimal medium at 37°C until the A 600 values of the cultures were 1.0. Then, 250 nmol of 14 C-labeled spermidine (GE Healthcare) was added to each culture, and they were then incubated for an additional 10 h. Samples of the cells were treated with 0.5 mM H 2 O 2 for 30 min, collected by centrifugation, mixed with SDS-loading buffer containing 0 or 50 mM 2-mercaptoethanol (2-ME), and then boiled for 15 min. After removing cell debris by centrifugation, the samples were subjected to SDS-PAGE. The separated proteins were stained with Coomassie Brilliant Blue or detected using a PhosphorImager system BAS-1500 (Fuji, Tokyo, Japan).
In Vitro and in Vivo Determination of Gsp Levels after H 2 O 2 Treatment-For the in vitro assay, a 1.8 nM GspSA solution was first incubated with 500 M H 2 O 2 for 5 min to inactivate the GspSA amidase activity and then treated with catalase (final concentration 1 mg/ml) for 20 min to remove any remaining H 2 O 2 . Omission of H 2 O 2 served as the control. The samples were added to 250 mM Tris-HCl (pH 7.3), 2 mM GSH, 2 mM Spd, 1 mM ATP, 4 mM phosphoenolpyruvate, 10 mM MgCl 2 , and 5 units/ml pyruvate kinase. Aliquots of 100 l were withdrawn periodically and heated at 95°C for 10 min to inactivate the enzyme activities. Samples were then derivatized with mBBr, and Gsp and GSH contents were analyzed by HPLC (12).
For the in vivo assay, E. coli BL21(DE3) was cultured in M9 minimal medium until its A 600 was 1.5. Then the culture was treated with 1, 5, or 10 mM H 2 O 2 . After 10 min, the cells were centrifuged and lyophilized. Cells of suitable dry weight were lysed, and 800 M 2-ME (final concentration) was added. The lysates were derivatized with mBBr and then centrifuged at 8000 ϫ g for 30 min to remove cell debris. The resulting samples were subjected to HPLC to identify and quantify cellular thiol levels.
GspSA Conversion of Gsp-disulfide to GSH by a Gsp Amidase/GSH Reductase Couple-To determine if Gsp amidase could catalyze the hydrolysis of (GspS) 2 , 5 mM Gsp-disulfide and 10 M GspSA (1 g) were incubated at 37°C and then subjected to MALDI-TOF-MS. Similarly, to determine if (GspS) 2 could be converted to GSH by the Gsp amidase/GSH reductase couple, 0.23 unit/ml of GSH reductase (final concentration) was added to mixtures of 100 mM Tris-HCl (pH 7.3), 0.5 mM NADPH, 600 M (GspS) 2 at 25°C with or without 28.7 M GspSA. NADPH consumption was measured by monitoring fluorescence emission at 460 nm (excitation at 340 nm). Replacement of (GspS) 2 with oxidized GSH (GSSG, the native substrate of GSH reductase) served as the positive control, whereas the negative control did not include (GspS) 2 and GSSG. 14 C-labeled spermidine (Spd*) was added to cultures of NR754 (wild type) and HA61002 (⌬gspSA). NR754 has an endogenous GspSA that can conjugate GSH and Spd* to form 14 C-labeled Gsp (Gsp*; Fig. 1A). After uptake of Spd*, Gsp*-labeled proteins were detected in lysates of NR754 by phosphorimaging after SDS-PAGE (Fig. 1B, right). The proteins were Gsp S-thiolated was confirmed by treatment of the Gsp*-labeled cell lysates with 2-ME ( Fig. 1B, right), which decreased the amount of radiolabeled proteins observed and was attributed to disulfide cleavage by 2-ME. Proteins of HA61002 (⌬gspSA) were not radiolabeled because Gsp could not be synthesized in HA61002 (Fig. 1B, right). Interestingly, the enhanced level of GspSSPs after H 2 O 2 treatment was reminiscent of the protective effects offered by GSH S-thiolation (13), which implied that Gsp might also protect protein thiols against irreversible oxidation. Various Gsp-protein conjugates were formed (Fig. 1B, right), suggesting that Gsp S-thiolation of proteins may be involved in diverse types of biological processes.

Identification of E. coli Gsp S-Thiolated Proteins-To
Rapid Accumulation of Gsp in Vivo in the Presence of H 2 O 2 -To measure the intracellular Gsp level after treatment with H 2 O 2 , M9 minimal medium cultures of E. coli BL21(DE3) that were in stationary phase (A 600 ϭ 1.2) were treated with 1, 5, or 10 mM H 2 O 2 and then incubated for an additional 10 min. The cells were collected by centrifugation and then lysed. Monobromobimane (mBBr) was added into the lysates to derivatize thiol-containing compounds (12), and after HPLC analysis, mBBr-modified Gsp in the eluates was quantified (fluorescence emission at 480 nm, excitation at 375 nm). The addition of 1 or 10 mM H 2 O 2 led to a 2.2-or 3.6fold increase in Gsp concentration, respectively, within 10 min ( Fig   same conditions (Fig. 3B). The finding that H 2 O 2 affects the two activities differently is supported by the structural information described below.
To determine whether H 2 O 2 -mediated amidase inactivation could be rescued by a biological reducing agent, inactivated GspSA (treated with 500 M H 2 O 2 for 5 min and then incubated with catalase for 20 min to remove excess H 2 O 2 ) was exposed to GSH. The addition of 4 mM GSH (physiological concentration) recovered ϳ80% of the amidase activity (supplemental Fig. S2). To examine how selectively inactivated Gsp amidase affected the level of Gsp, GspSA was treated with GSH, Spd, ATP, phosphoenolpyruvate, and pyruvate kinase with or without prior exposure to H 2 O 2 . The mixtures were then treated with mBBr and analyzed by HPLC. Whether H 2 O 2 -treated or not, the Gsp concentrations initially increased and then declined (supplemental Fig. S3); therefore, the level of Gsp was tightly regulated. After pretreatment with H 2 O 2 , Gsp accumulated to a greater extent than it did otherwise (no treatment). Additionally, less GSH was present at all times longer than the initial stage (ϳ25 min). Both of these observations suggested that H 2 O 2 selectively inactivates Gsp amidase, but the amidase activity recovers at a later stage. More Gsp was produced as a consequence of oxidative damage.
Identification of Cys 59 -Sulfenic Acid by X-ray Crystallography and a Chemical Modification Study-The structure of the H 2 O 2 -treated Gsp amidase fragment (GspAF_H 2 O 2 ; supplemental Table S1) was obtained by soaking a GspAF crystal in crystallization buffer containing 1 mM H 2 O 2 for 3 days before x-ray crystallography. The electron density map of GspAF_ H 2 O 2 (Protein Data Bank entry 3A2Z) at 1.5 Å resolution has a density not seen in untreated GspAF that extends from Cys 59 S ␥ and corresponds to two adjacent oxygen atoms (Fig. 4A). The distance between the S and O1 is 2.1 Å, the C-S-O1 angle is 96.0°, and the S-O1-O2 angle is 94.2°. The second oxygen atom (O2) is unusually close to O1 (2.2 Å). Using the structural and other information (see below), we suggest that the Cys 59 thiol was oxidized to sulfenic acid and that its oxygen atom forms a very short hydrogen bond (Ͻ2.3 Å) (14) with a water molecule (H 2 O triad ; Fig. 4B and supplemental Table S1); therefore, the extra electron density probably arose as a consequence of an S-O1⅐⅐⅐H⅐⅐⅐O2-H hydrogen bond. Partial negative charges could be present at O1 and O2 because such hydrogen bonds have partial ionic character. Conversely, we do not believe that a hydroperoxide-derivatized cysteine (-S-O-O-H) was present because the S-O and O-O distances are not compatible with such a compound, and it would be less stable than a sulfenic acid (15).
The sulfenic acid that was produced when a GspAF crystal was soaked with 1 mM H 2 O 2 for 20 min was reduced to a free thiol when subsequently treated with 1 mM GSH (data not shown). Interestingly, the sulfenic acid was not further oxidized by either prolonged incubation with H 2 O 2 or by a greater concentration of H 2 O 2 (10 mM). To our surprise, Cys 59 was covalently bonded to an acetate oxygen in the GspAF_acetate crystal structure (Protein Data Bank code 3A30; supplemental Fig. S4). This S-modification was probably caused by a nucleophilic attack of an acetate ion on the sulfenic acid, which was favorable because of the large acetate concentration (100 mM) and the prolonged exposure.
Although Cys 59 , which is the catalytic nucleophile for GspSA amidase, was oxidized by H 2 O 2 , other GspSA cysteines, including Cys 338 and Cys 539 , which form part of the synthetase binding site (9), were not oxidized when crystals of GspSA were treated with H 2 O 2 (data not shown).
To further corroborate that the Cys 59 thiol was oxidized to a sulfenic acid (16), GspAF was first treated with 100 M H 2 O 2 for 10 min, which fully inactivated GspAF, and then reacted with 100 M 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which specifically reacts with sulfenic acid (16), at 25°C for 12 h. After removing excess reagents, derivatized GspAF was digested with trypsin, and its hydrolytic products were analyzed by MALDI-TOF-MS (Fig. 4C). The signal (M ϩ H) ϩ at m/z 1176.8 is expected for the adduct formed by the GspSA sequence 57 WQCVEFAR 64 (M r ϭ 1037) and dimedone (M r ϭ 140). The presence of b-and y-series ions confirmed that dimedone modified the Cys 59 sulfenic acid. Therefore, the thiol of Cys 59 was oxidized to the sulfenic acid by H 2 O 2 . . Synthetase activity was measured as the consumption of NADH by the pyruvate kinase/lactate dehydrogenase couple, which also used ADP generated by Gsp synthetase.

Hydrolysis of Gsp-disulfide and Gsp S-Thiolated Proteins and Peptides by Gsp
Amidase-E. coli lacks an enzyme that can reduce (GspS) 2 (17). If (GspS) 2 is involved in E. coli redox regulation, then there must be a way to convert (GspS) 2 to Gsp. We next showed that it is possible to convert (GspS) 2 to GSH by coupling Gsp amidase and GSH reductase activities. (GspS) 2 (Fig. 5A). A second assay was per-formed to determine if (GspS) 2 was a substrate for GSH reductase. GSH reductase activity was detected as NADPH consumption, which was monitored by NADPH absorption at 340 nm (Fig. 5B). GSH reductase alone (without the addition of GSSG) served as the negative control (open circles in Fig. 5B). No activity was observed when (GspS) 2 was incubated with only GSH reductase and NADPH (filled squares), in contrast to the result found for GSSG (filled circles). Activity was also observed when (GspS) 2 was treated with GspSA and GSH reductase (open triangles). The experimental results presented in Fig. 5B suggested that both (GspS) 2 and Gsp-SG were converted to GSH and Spd via consecutive reactions by Gsp amidase and GSH reductase (Fig. 5C). We next tested whether Gsp S-thiolated peptides and proteins were substrates for Gsp amidase. Lysates of E. coli proteins were treated with disulfide of biotinated Gsp ((Biotin-GspS) 2 ) (Fig. 6A) to generate biotin-labeled GspSSPs (biotin-GspSSPs), which could be detected with an antibiotin antibody (Fig. 6B). Treatment of these proteins with GspSA removed Spd-biotin moieties because biotin-GspSSPs were no longer observed after staining with the anti-biotin antibody (Fig. 6C, left panel, lane 3). Treatment with 2-ME also produced the same result, presumably because 2-ME cleaved Gsp-protein disulfides (Fig. 6C, left panel, lane 2). A synthetic Gsp-containing peptide (T28-Gsp) was also incubated with GspSA, and a MALDI-TOF-MS spectrum of that incubation mixture indicated that T28-Gsp was a substrate for Gsp amidase (supplemental Fig. S5). Therefore, Gsp amidase can hydrolyze a variety of Gsp-derivatized substrates, yielding Spd and GSH S-thiolated proteins/peptides.
Sensitivities of Different E. coli Strains to Oxidative Stress-To characterize how Gsp and GspSA protect against oxidative stress, cultures of NR754 (wild type) and HA61002 (⌬gspSA) were treated with various concentrations of H 2 O 2 , and the viabilities of the cells were examined after treatment. The strains were cultured in M9 minimal medium until the cells reached mid-exponential phases, at which point they were treated with H 2 O 2 , incubated for 1 h, serially diluted with PBS buffer, and then transferred to LB plates. The viabilities of NR754 and HA61002 under oxidative stress were similar (supplemental Fig. S6), which is consistent with a previous report that an E. coli strain lacking the ability to synthesize GSH was as resistant to H 2 O 2 as the corresponding wild-type strain was (18).
Because glutaredoxins can reduce GSH S-thiolated proteins (GSSPs) (19), we wanted to determine if these enzymes are part of a redox cycle involving Gsp S-thiolation. The viabilities of two glutaredoxin null strains (⌬grxA and ⌬grxB) and two double mutant strains (⌬grxA⌬gspSA and ⌬grxB⌬gspSA) were examined after they were treated with H 2 O 2 . When preincubated with 1 or 5 mM H 2 O 2 for 1 h, ⌬grxA and ⌬grxB had survivability values of 60 and 30%, respectively (Fig. 7). The double mutant strains were even more susceptible to oxidative damage because their survivabilities were reduced by 70 and 99% after exposure to 1 and 5 mM H 2 O 2 , respectively. Therefore, there is a synergistic protective effect by GspSA and Grx against oxidative damage. Moreover, in a separate experiment, the levels of GspSSPs were substantially decreased when recombinant glutaredoxin (Grx1 or Grx2) was added to E. coli FIGURE 5. Conversion of (GspS) 2 to GSH and Spd by the Gsp amidase/GSH reductase couple. A, a MALDI-TOF spectrum that shows the molecular weights of the hydrolysis products after (GspS) 2 was treated with GspSA for 0 min (top), 10 min (middle), or 4 h (bottom). GSSG was the end product. B, the Gsp amidase/GSH reductase coupled assay. (GspS) 2 cannot be reduced by GSH reductase alone (f), but it is reduced when both GspSA and GSH reductase are present (‚). GSSG plus GSH reductase served as the positive control (F). GSH reductase alone served as the negative control (E). C, reaction scheme for the enzymatic conversion of (GspS) 2 to GSH and Spd by the Gsp amidase/GSH reductase couple.
lysates that contained Gsp*SSPs (supplemental Fig. S7). These results strengthen the idea that Grxs participate in the reduction of Gsp S-thiolated proteins.

Redox Regulation of Gsp and Gsp S-Thiolated Proteins-
Most organisms use GSH to regulate their intracellular thiol and disulfide levels. Gsp is a GSH derivative found only in some bacteria and parasitic protozoa. As reported herein, we showed that Gsp forms mixed disulfides with the thiols of E. coli proteins in vivo and that the amounts of these Gsp derivatives are linked to intracellular redox conditions. In vivo Gsp S-thiolation of E. coli proteins is affected by several factors, including the intracellular Gsp concentration. We demonstrated that the Gsp level increases when E. coli is oxidatively stressed. The selective inactivation of Gsp amidase provides a rational explanation for this event. When E. coli is exposed to reactive oxygen species, the thiol of Cys 59 of the active-site amidase domain is oxidized to sulfenic acid, which inactivates Gsp amidase and consequently causes Gsp to accumulate (Fig. 8). Accumulated Gsp possibly scavenges oxidants directly or forms mixed disulfides with protein thiols. Once the source of the oxidative stress has been eliminated, Gsp amidase activity can be rescued by reaction of the sulfenic acid with a reducing reagent (e.g. GSH or Gsp). Sulfenic acid is a reactive electrophile and reacts with thiol reagents, such as GSH, to generate mixed disulfides. The amidase active site is solvent-exposed, which should allow GSH or other small thiol-containing molecules to react with the sulfenic acid. After formation of a mixed disulfide, an additional thiol exchange may continue regenerating the thiol of Cys 59 and thereby recovering amidase activity (supplemental Fig. S8). The reactivated amidase may then hydrolyze either excessive Gsp to GSH and Spd and/or GspSSPs to GSSPs and Spd. Gsp could also be oxidized to produce (GspS) 2 and/or other mixed disulfides upon reaction with reactive oxygens. We showed that GSH reductase alone cannot reduce (GspS) 2 but that Gsp amidase must first remove the spermidine moiety. The conversion of (GspS) 2 to Spd and GSH by the Gsp amidase/GSH reductase couple suggests that Gsp may be part of an oxidative defense mechanism that does not require a Gspspecific reductase. Such a reductase has not been found in E. coli (17). The results allow us to propose a reaction scheme (Fig. 8) that explains how Gsp amidase activity is modulated in vivo so that the Gsp level reflects the intracellular redox condition.
Glutaredoxins reduce mixed disulfide bonds between GSH and GSSPs by forming a GrxSSG intermediate that can be subsequently reduced by a molecule of GSH (20). The protein expression level of Grx1 is enhanced under oxidative stress in E. coli, whereas Grx2 is constitutively produced (21). We found that the amounts of GspSSPs decreased in the presence of Grx1 or Grx2 (supplemental Fig. S7), suggesting that Grxs can reduce mixed disulfides. As mentioned, Gsp amidase catalyzes the hydrolytic removal of Spd from GspSSPs, leading to the formation of GSSPs. A Grx can then reduce the GSSPs to generate free protein thiol(s) (supplemental Fig. S9). However, we cannot exclude the possibility that glutaredoxin directly reduces GspSSPs, with the resulting Grx-Gsp mixed disulfide intermediate subsequently reduced via thioldisulfide exchange (supplemental Fig. S9). Because ⌬grxA⌬gspSA and ⌬grxB⌬gspSA were more easily killed by H 2 O 2 than were ⌬grxA and ⌬grxB, it appears that the activities of Grx and GspSA can provide a coordinated defense against oxidative damage in E. coli.
To date, most studies concerning protein S-thiolation have focused on GSH, because GSH is abundant in cells (4). However, protein S-thiolation by other thiol-containing molecules, such as cysteine or Gsp, may have different effects. For example, a previous study demonstrated that GSH S-thiolation of Ca 2ϩ -dependent protein kinase C␣ inhibits the activity of Ca 2ϩ -dependent protein kinase C␣ and its isozymes, whereas cysteine S-thiolation does not (22). Both trypanothione (TSH) and Gsp are more efficient reducing agents than is GSH. The non-enzymatic reductions of dehydroascorbate and H 2 O 2 by Gsp and TSH are several times faster than those by GSH (23,24). It has also been found that Gsp was a more efficient S-thiolating agent than was GSH (25). GSH is negatively charged (Ϫ1) at physiological pH, whereas Gsp is positively charged (ϩ2). Gsp and GSH therefore introduce opposite charges into the proteins that they thiolate. Protein thiols are deprotonated to form thiolates that interact more readily with (GspS) 2 at pH Ͼ6, rather than GSSG (25). Although GSH and Gsp have distinct physical and chemical properties, it is still difficult to elucidate how GSH and Gsp function in vivo because of the involvement of the GspSA amidase-catalyzed conversion of GspSSPs to GSSPs.
Dissimilar Amidase Active Sites Linked to Differential Redox Regulation-Parasitic protozoa use TSH, which is synthesized by TSH synthetase/amidase to defend against oxidative damage. Like GspSA, TSH synthetase/amidase has an amidase activity that can hydrolyze the amide bond of Gsp. Therefore, it would be interesting to know if TSH amidase can be selectively and reversibly inactivated.
In the x-ray structure of Leishmania major TSH synthetase/amidase, the C-terminal segment partially obstructs accessibility to the catalytic Cys 59 nucleophile (26). Consequently, the active site of TSH amidase is substantially blocked, in contrast with that of GspSA, which is part of a large, solvent-accessible cleft. Even if the TSH amidase Cys 59 thiol could be oxidized to sulfenic acid and/or form a mixed disulfide with GSH or TSH, the restricted access almost certainly would impede subsequent thiol exchange (supplemental Figs. S8 and S10). Because access to the TSH amidase active site is partially obscured, TSH reductase and TSH tryparedoxin may compensate as a defense mechanism against reactive oxygen in parasitic protozoa. It is also possible that, with the evolutionary emergence of TSH reductase and TSH tryparedoxin, TSH amidase degenerates and no longer participates in redox regulation.  coli. When exposed to reactive oxygen species (ROS), the active-site Cys 59 thiol of Gsp amidase is oxidized to sulfenic acid (1), which causes the inactivation of Gsp amidase (ϫ) (2). Because Gsp synthetase is not affected by ROS, intracellular Gsp accumulates (3). Gsp may then scavenge harmful oxidants by forming Gsp-disulfides and other small molecule disulfide compounds (4A) and/or protecting protein thiols from oxidation by Gsp S-thiolation (4B). With the removal of the oxidative stresses, intracellular GSH and/or Gsp may rescue oxidized Gsp amidase, restoring amidase activity (4C). Reactivated Gsp amidase may hydrolyze Gsp to GSH and Spd or hydrolytically remove Spd from Gsp-disulfide or Gsp-modified proteins. Finally, the amount of Gsp returns to its basal level (5).