Methionine sulfoxide reductase A is a stereospecific methionine oxidase

Abstract

Methionine sulfoxide reductase A (MsrA) catalyzes the reduction of methionine sulfoxide to methionine and is specific for the S epimer of methionine sulfoxide. The enzyme participates in defense against oxidative stresses by reducing methionine sulfoxide residues in proteins back to methionine. Because oxidation of methionine residues is reversible, this covalent modification could also function as a mechanism for cellular regulation, provided there exists a stereospecific methionine oxidase. We show that MsrA itself is a stereospecific methionine oxidase, producing S-methionine sulfoxide as its product. MsrA catalyzes its own autooxidation as well as oxidation of free methionine and methionine residues in peptides and proteins. When functioning as a reductase, MsrA fully reverses the oxidations which it catalyzes.

Methionine is an essential amino acid for most mammals including humans. Approximately 99% of cellular methionine is found in protein, with the remainder in small molecules such as free methionine and S-adenosyl-methionine (1). Methionine, as cysteine (Cys), the other sulfur-containing amino acid, is relatively easily oxidized to yield methionine sulfoxide. Oxidation by hydrogen peroxide, hypochlorous acid, and other reactive species creates a chiral center at the sulfur so that the methionine sulfoxide (MetO) produced is a mixture of the S and R epimers (2). Reduction of the sulfoxide back to methionine is catalyzed by methionine sulfoxide reductases (Msr), enzymes found in almost all organisms from microbes to humans. Two distinct classes of reductases are required, one to reduce the S epimer [methionine sulfoxide reductase A (MsrA)] and one to reduce the R epimer [methionine sulfoxide reductase B (MsrB)]. MsrA was described many years before MsrB and as a consequence most studies focused on MsrA. Although the enzyme is almost ubiquitous and a variety of studies establish its importance, its actual roles in cellular function remain unclear. Knocking out MsrA caused increased susceptibility to oxidative stress in mice (3), yeast (4), and bacteria (5–7). Conversely, overexpressing MsrA conferred increased resistance to Drosophila (8), Saccharomyces (9), Arabidopsis (10), PC-12 cells (11), and human T cells (9). Overexpression in Drosophila doubled the lifespan of the flies (8).

There is reasonable evidence that cyclic oxidation and reduction of methionine residues in proteins act to scavenge reactive oxygen and nitrogen species (12). Methionine residues can thus function as “macromolecular bodyguards,” preventing the oxidation of critical residues in structural proteins and enzymes (13). Because methionine oxidation is a reversible covalent modification, investigators have frequently suggested that it may have a role in cellular signaling and regulation but the experimental evidence to support such a role is limited (14–16). Although MsrA is a stereospecific MetO reductase, no stereospecific methionine oxidase has yet been described. If such an oxidase exists, it would complete the enzymatic requirements for a reversible covalent modification. We now show that MsrA is a dual-function enzyme with the ability to stereospecifically catalyze both the oxidation of methionine in proteins to S-MetO as well as reduction of S-MetO back to methionine.

Results

Stoichiometry.

Branlant and coworkers have studied in careful detail the mechanism of the MsrA reaction in bacteria (17, 18). In the absence of reducing agents, each molecule of MsrA reduces two molecules of MetO. Reduction of the first MetO generates a sulfenic acid at the active site cysteine, and it is reduced back to the thiol by a fast reaction, which generates a disulfide bond in the resolving domain of the protein. The second MetO is then reduced and again generates a sulfenic acid at the active site. Because the resolving domain cysteines have already formed a disulfide, no further reaction forms. The mass of the protein has increased by 14 Da, +16 Da from the sulfenic oxygen and -2 Da from the disulfide. When the mouse MsrA was incubated with MetO for 30 min, the stoichiometry of the Met formation appeared at first examination to be identical to that for the bacterial enzymes (Fig. 1A). However, when the incubation time was lengthened, it became clear that three MetO are reduced by each MsrA (Fig. 1B) and that the mass of protein increased by 12 Da, not 14 Da (from 23,648.6 Da to 23,660.6 Da, with an error of ∼0.5 Da for each mass). We therefore measured the mass change of MsrA during the reaction and identified four major species (Fig. 1C).

Fig. 1.

Met formation and MsrA mass changes. (A) Met production as a function of MetO concentration. Wild-type MsrA (1 μM) was incubated in 50 mM sodium phosphate, 1 mM DTPA, pH 7.5 without reductant at 37 °C for 30 min. (B) Met production as a function of time. The incubation was as in A with MetO held at 50 μM. (C) Mass analysis from the incubations shown in B. Minus 2 Da species, ◊; -4 Da species, Δ; +12 Da species, ∇.

Characterization of the MsrA Species.

The species with different masses could be purified by taking samples at different times of incubation with MetO and purifiying them by reverse phase chromatography (Fig. S1). These were treated with iodoacetamide to alkylate cysteine thiols, digested with trypsin, and the peptides mapped by reverse phase HPLC and tandem mass spectrometry. Peptides that were modified during the reaction with MetO were identified and their posttranslational modifications established (Table 1). Similar to the bacterial MsrA, the reduction of the first MetO of the mouse MsrA leads to disulfide formation in the resolving domain, with a net mass change of -2 Da. The second MetO further decreases the mass to -4 Da from the native, a distinct difference from the +14 Da observed in the bacterial form. Although the additional decrease of 2 Da could have resulted from the formation of a second disulfide bond, it did not. We then considered that a sulfenic acid did form on the active site Cys72 but then reacted with the nitrogen of an adjacent residue’s peptide bond to form a sulfenamide, a well-documented reaction of protein sulfenic acids (19, 20). Sulfenamide formation would generate a modified protein of -4 Da from the native, and because the electrospray ionization technique is dehydrating, sulfenamide formation would be favored in the mass spectrometer. Dimedone and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) both react with sulfenic acid to give a stable derivative. NBD reacted with the unoxidized Cys of each form. Dimedone did not react with the native nor -2 Da forms. Both dimedone and NBD reacted with the -4 Da form to give the derivative expected from a sulfenic acid (Table 1). Thus, except for the secondary formation of a sulfenamide by the mouse MsrA, its catalytic cycle thus far was the same as that observed for the bacterial enzyme.

Table 1.

Characterization of MsrA species

MsrA species, calculated mass without reagent	Intact, 23,648.6 Da		-2 Da, 23,646.6 Da		-4 Da, 23,644.6 Da		+12 Da, 23,660.6 Da
	NBD	Dimedone	NBD	Dimedone	NBD	Dimedone	NBD	Dimedone
Observed mass	24,301.1	23,648.5	23,973.1	23,646.4	23,988.7	23,784.7	24,004.4	23,800.8
Observed mass change	+652.5	−0.1	+324.5	−2.2	+340.1	+136.1	+355.8	+152.2
Calculated mass change	+652.0	0.0	+324.2	−2.0	+340.2	+136.2	+356.2	+152.2
Assignment*	4 Cys-S-NBD	—	1 Cys-S-S-Cys, 2 Cys-S-NBD	1 Cys-S-S-Cys	1 Cys-S-S-Cys,1 Cys-S-NBD, 1 Cys-S-O-NBD	1 Cys-S-S-Cys, 1 Cys-S-Dimedone	1 Cys-S-S-Cys, 1 Cys-S-NBD, 1 Cys-S-O-NBD, 1 MetO	1 Cys-S-S-Cys, 1 Cys-S-Dimedone, 1 MetO

^*For clarity, the status of Cys107 is not shown in the diagram in the next row.

Reduction of the third MetO generated an MsrA species which was +12 Da heavier than the native form. Because this species was +16 Da from the -4 Da species, it probably resulted from the addition of an oxygen to the -4 Da form. The most likely explanation would be the addition of another oxygen to the active site Cys72, generating either a sulfinic acid or a more complex derivative such as an S-oxide (21). However, the +12 Da form still reacted with dimedone and NBD to give sulfenic acid derivatives. In addition, the +12 Da form as well as the -2 Da and -4 Da forms was converted back to the native mass upon addition of a reducing agent such as dithiothreitol or tris(2-carboxyethyl)phosphine (TCEP).

These characteristics indicated that the oxygen was on a residue other than the active site Cys72. Peptide mapping and sequencing by mass spectrometry demonstrated that the modification giving rise to the +12 Da form was conversion of Met229 to methionine sulfoxide, resulting in a 16 Da increase in the mass of the tryptic peptide (Fig. 2 and Table S1). Thus, the mouse MsrA can autooxidize a methionine residue. After reduction of the second MetO, the sulfenic acid on Cys72 oxidizes Met229, and is itself reduced back to a thiol which allows it to react with a third MetO. Oxidation of Met229 is relatively slow (Fig. 1B).

Fig. 2.

Covalent modifications in the C-terminal residues of MsrA. Unmodified and the +12 Da forms were mapped. Oxidation of Met229 to the sulfoxide was confirmed by sequencing, which also confirmed that the two cysteine residues in peptides 1 and 2 were alkylated while they formed a disulfide in peptides 3 and 4.

When wild-type MsrA was incubated with Met¹⁶O in H₂¹⁶O, the protein mass increased by 12 Da. When incubated with Met¹⁶O in H₂¹⁸O, the mass increased by 14 Da (Table 2). Thus the oxygen in Met₂₂₉O is not transferred from the substrate MetO but is derived from water. When the +12 Da form was incubated with NBD, the mass of the derivative was 4 Da higher when carried out in H₂¹⁸O rather than H₂¹⁶O, demonstrating that both the oxygen of Met₂₂₉O and that of the sulfenic acid on Cys72 are from water. To rule out possible complexities arising from disulfide formation between Cys218 and Cys227 and sulfoxide formation on Met229 in wild-type MsrA, we constructed a mutant MsrA in which formation of sulfenic acid should be the only covalent modification induced by exposure to substrate. Cys107, Cys218, and Cys227 were mutated to Ser and the six C-terminal residues were deleted (C107S/C218S/C227S/Δ228–233). When incubated with MetO, its mass decreased by 2 Da, consistent with sulfenic acid formation followed by conversion to the sulfenamide (Table 2). Addition of NBD confirmed the presence of sulfenic acid. When the latter reaction was carried out in H₂¹⁸O, the NBD derivative was 2 Da heavier than when performed in H₂¹⁶O, confirming that the oxygen of the sulfenic acid is donated by water.

Table 2.

The oxygen of Cys₇₂sulfenic acid and of Met₂₂₉O is derived from water

MsrA species, calculated mass	Wild-type, 23,648.6 Da		C107S/C218S/C227S/Δ228–233, 22,931.5 Da
	NBD		NBD
	−	+	−	+
In H216O Observed mass Change in mass	23,660.6+12.0	24,004.6+356.0	22,929.9−1.6	23,110.3+178.8
In H218O Observed mass Change in mass	23,662.5+13.9	24,008.3+359.7	22,929.9−1.6	23,112.3+180.8
Difference between H218O and H216O	+1.9	+3.7	0.0	+2.0
Assignment*	1 Cys-S-S-Cys, 1 Cys-S-†OH, 1 Met†O		1 Cys-S-†OH

^*For clarity, the status of Cys107 is not shown in the diagram in the next row.

^†Oxygen incorporated from water. Two micrograms of MsrA were lyophilized, redissolved in 50 μL H₂¹⁶O or H₂¹⁸O containing 10 mM l-MetO, incubated for 30 min at 37 °C, and then incubated another 60 min with or without 0.5 mM dimedone or NBD.

Methionine Oxidation is Stereospecific for the S Epimer.

Oxidation of Met by chemical oxidants such as hydrogen peroxide generates a mixture of the S and R epimers (22). MsrA is able to reduce only the S form and MsrB can reduce only the R form (23, 24). Reverse phase chromatography of o-phthalaldehyde derivatized MetO provides a convenient assay for the S and R epimers (23) because the more hydrophilic S epimer elutes earlier than the R epimer (Fig. 3A, Upper). When MetO produced by peroxide oxidation of Met was incubated with MsrA and DTT, only the S epimer was reduced (Fig. 3A, Lower). When a sulfenic acid form of MsrA was incubated with Met in the absence of DTT, only S-MetO was produced (Fig. 3B, Upper). Addition of DTT supported reduction back to Met (Fig. 3B, Lower).

Fig. 3.

MsrA is stereospecific for the S epimer. (A) Reduction. A mixture of the S and R epimers of MetO was prepared by incubation of 500 μM l-Met with 1 M hydrogen peroxide for 2 h at 37 °C. The Upper tracing shows the chromatogram of the untreated product. The Lower tracing shows that only the S epimer was reduced when 20 μM l-MetO was incubated with MsrA and 10 mM DTT. (B) Oxidation. 20 μM of the sulfenic acid form of MsrA (C107S/C218S/C227S/Δ228–233) was incubated with 20 μM l-Met for 6 h at 37 °C. The product is shown in the Upper tracing, and its complete reduction upon addition of 10 mM DTT in the Lower tracing. For both A and B, incubation with 10 mM DTT alone had no effect.

In addition to free Met and Met229 of MsrA, we tested two other peptides and the natively unfolded protein α-synuclein as substrates (Table 3). The tripeptide Gly-Met-Gly and the hexapeptide Pro-Met-Ala-Ile-Lys-Lys were both substrates. The hexapeptide sequence is that of the C terminus of MsrA. In addition to the Met form of the peptide, we also synthesized the MetO form. They were used as substrates for the wild-type MsrA to determine kinetic parameters for the oxidation and reduction reactions. From two separate determinations, the K_m for the reduction of the S-MetO-containing peptide was 0.12 mM and the V_max was 1.4 μmol·min^-1mg^-1protein. In the oxidation reaction, the K_m was 3.5 mM and the V_max was 0.25μmol· min^-1mg^-1 protein. α-Synuclein was oxidized at both Met1 and Met5 but not at Met116 or Met127. Addition of DTT allowed MsrA to completely reduce the MetO it produced, confirming that oxidation of Met in peptide linkage is also stereospecific.

Table 3.

Substrates of the MsrA oxidase

Substrate	Unoxidized mass*	Oxidized mass*	Change in mass	Assignment
Gly-Met-Gly	263.09	279.09	+16.0	Met2O
Pro-Met-Ala-Ile-Lys-Lys	686.41	702.41	+16.0	Met2O
C72A MsrA	23,616.6	23,631.2	+14.6	Met229O Cys218S-SCys227
α-Synuclein	14,460.2	14,476.4, 14,492.8	+16.2, +32.6	Met1O, Met1O, and Met5O

^*The measured masses are monoisotopic for the peptides and average for the proteins. The Met residues which were oxidized were determined by tandem mass spectrometric analysis of the peptides and of tryptic digests of the proteins.

The bacterial MsrA oxidized the hexapeptide and α-synuclein although the rate of oxidation was much slower than that catalyzed by mouse MsrA. The bacterial MsrB and free methionine-R-sulfoxide reductase did not undergo autooxidation nor did they oxidize the hexapeptide or α-synuclein. Thus, of the enzymes tested, only MsrA can function as a stereospecific methionine oxidase. There is a Val rather than Met at position 231 in human MsrA and no Met in the C-terminal region of Escherichia coli MsrA, and neither enzyme had significant autooxidation (Table 4). Introduction of a Met in the C-terminal region rendered both human and E. coli MsrA autooxidizable.

Table 4.

Ability of recombinant MsrA to autooxidize Met residues

	Mouse		Human		E. coli
	Wild-type	M229L	Wild-type	V231M	Wild-type	P209M
Autooxidation	Yes	No	No	Yes	No	Yes

Discussion

Methionine and cysteine are the two sulfur-containing amino acids present in proteins. Well-known roles of cysteine include catalysis, structure, redox sensing and regulation, and antioxidant defense. We have noted that methionine residues share at least two functions with their sulfur-containing sibling: antioxidant defense and regulation of cellular function through reversible oxidation and reduction (25). Although cysteine is well-recognized for the ease of its oxidation, it is often not appreciated that methionine is quite readily oxidized to MetO (22, 26). Conversion of methionine to its sulfoxide is mediated by a host of reactive species over a broad pH range. For example, hydrogen peroxide readily reacts with certain methionine residues in proteins even at pH 5 or lower. Cysteine residues are typically quite resistant to oxidation under these conditions because the thiol of cysteine must be ionized for oxidation to occur, and the pK_a of cysteine is over eight unless altered by other residues within the protein. Although nonenzymatic oxidation of methionine could drive one half of a reversible covalent modification cycle, the product would be a mixture of the S and R epimers of MetO. Reversal would require two enzymes, either MsrA and MsrB or one reductase and an epimerase (which has not yet been described). Although such a mechanism seems possible, the requirement for two separate enzymes to reverse the oxidation is not appealing. A stereospecific methionine oxidase would obviate that problem. Although flavin monooxygenase 3 stereospecifically oxidizes free Met to R-MetO (27), it does not oxidize methionine residues whose amino group is in peptide linkage (28).

In the course of an investigation of the catalytic cycle of the mouse MsrA we discovered that the enzyme mediated autooxidation of its Met229, analogous to the autophosphorylation mediated by many protein kinases. Unlike the kinases, a second enzyme is not required to reverse the covalent modification. MsrA functions as both a stereospecific methionine oxidase and a stereospecific methionine sulfoxide reductase. Dual-function mediators of reversible covalent modification in proteins have been known for many years. For example, bacterial glutamine synthetase, a key enzyme in intermediary metabolism, is regulated by reversible adenylylation of a tyrosine residue (29). It was initially thought that adenylylation and deadenylylation were mediated by separate enzymes, but both reactions are catalyzed by the same protein, initially named P_I (30). No futile cycle occurs because the direction of the reaction is regulated by a second protein, P_II, which interacts with P_I (31). We propose that MsrA mediates both the oxidation and reduction of methionine residues within certain proteins. The direction of the reaction would be controlled either by a reversible covalent modification of MsrA or, perhaps more likely, by interaction with another protein. The crystal and solution structures of the microbial and mammalian MsrAs reveal a common domain structure (32–34) and suggest a simple mechanism for regulation by protein–protein interaction (Fig. 4). In mouse MsrA, the active site is distant from the carboxyl domain which contains Cys218 and Cys227, which are required to reduce the sulfenic acid formed when MsrA acts as a reductase. They form a disulfide which in vivo is reduced by thioredoxin to complete the reductase cycle. In vitro, investigators typically replace the thioredoxin thioredoxin reductase system by a low molecular weight reductant such as DTT or TCEP. Both DTT and TCEP can directly reduce the active site sulfenic acid, obviating the need for the resolving cysteines in the carboxy domain. However, when MsrA was incubated with MetO in the absence of these reducing agents, it acted as a methionine oxidase. In vivo, a protein which binds to the carboxy domain could prevent the resolving cysteines from accessing the active site, thus allowing the oxidase reaction to proceed. The same functional result would be obtained if an interacting protein blocked the access of thioredoxin to the disulfide bond in the carboxy domain. MsrA could therefore mediate a regulated, reversible oxidation of methionine residues in proteins and provide a mechanism for control of cellular functions, including signaling. In our assays, the hexapeptide substrate’s kinetic parameters were an order of magnitude more favorable in the reduction direction than in the oxidation direction, but it is probably not appropriate to compare them. The reason is that the reduction reaction requires binding of only one substrate at the active site, the oxidized peptide. However, the oxidase reaction requires binding of MetO to the active site, oxidation of Cys72 to the sulfenic acid, release of Met, and finally binding of the reduced peptide to the active site. Identification of in vivo substrates of the MsrA oxidase and assessment of the role of their oxidative modification in cell signaling will be important in determining the physiological significance of the proposed mechanism.

Fig. 4.

Potential mechanism for regulation of the reductase and oxidase reactions. When thioredoxin has access to the carboxy-domain resolving cysteines, MsrA functions as a reductase. When access of thioredoxin is blocked as a consequence of a conformational change induced by a reversible covalent modification or, as shown, by the binding of a another protein, MsrA functions as an oxidase.

For experimental convenience we used only MetO to drive the oxidase reaction in these studies. Reactive species such as hydrogen peroxide, hypochlorous acid, chloramines, and peroxynitrite can also oxidize cysteine to the sulfenic acid (26). Hydrogen peroxide is well-recognized to participate in cell signaling through oxidation of active site thiols of enzymes (35). Hypochlorous acid and chloramines are relatively specific oxidizing agents for the sulfur-containing amino acids (36). In vitro, peroxynitrite readily oxidizes methionine residues to the sulfoxide (37), but it is questionable whether this reaction occurs in vivo (38). Whether these or other small reactive species act on MsrA in vivo remains to be investigated.

We suggest that the catalytic mechanism for oxidation of methionine is simply a reversal of that for reduction of methionine sulfoxide. Based on experimentally derived data, a detailed mechanism for the catalytic cycle of Msr acting as a reductase has been proposed by Branlant and coworkers (18). Both MsrA and MsrB are thought to form sulfurane transition state with bipyramidal geometry. A rearrangement of this transition state was hypothesized to occur, leading to formation of the sulfenic acid on the catalytic cysteine and release of the methionine product. In our current studies, when MsrA acted as a methionine oxidase, the sulfoxide oxygen was derived from water and not from the substrate methionine sulfoxide. Because the sulfenic acid of MsrA can form a sulfenamide, reversal of sulfenamide formation by hydrolysis would introduce an oxygen from water in the sulfenic acid. However, direct participation of water in the catalytic reaction is consistent with the relationship of the substrate and the catalytic cysteine seen in crystal structures of MsrA (32–34). The sulfoxide oxygen points away from the cysteine thiolate and is too far away for direct transfer to the thiolate. Instead, the thiolate attacks the sulfur of the methionine sulfoxide as part of a catalytic mechanism which requires participation of water as a reactant. One possible scheme is shown in Fig. 5.

Fig. 5.

Proposed mechanisms for the oxidase and reductase reactions. The reductase mechanism shown on the right is an extension of that proposed by Branlant and coworkers (18). In particular, we propose that the oxygen atom on the sulfenic acid is derived from a water molecule rather than methionine sulfoxide. At the initiation of the reductase reaction (II), the oxygen of the sulfoxide substrate is equidistant (2.6 Å) from the carboxylate oxygen of Glu115 and the hydroxyl of Tyr103 (II) (34). Glu115 acts as a proton donor to the sulfoxide, and the protonated hydroxyl sulfonium intermediate (III) is stabilized by hydrogen bonding with Tyr103. Nucleophilic attack of the hydroxyl sulfonium intermediate by the thiolate of Cys72 forms the sulfurane intermediate (IV) (42–44). It has been suggested that the sulfurane hydroxyl group could be protonated by a second methanthiol molecule and form the sulfonium intermediate upon loss of water. The pK_a of methanthiol and the hydroxyl of tyrosine are both about 10.2, leading us to suggest that Tyr103 is the second proton donor, facilitating formation of the sulfonium intermediate upon leaving of a water molecule which would be exchangeable with solvent water (V). Another water molecule, seen in several crystal structures of MsrA is sited 2.8 Å from Cys72 on the opposite side from the bound substrate. This location places it in perfect position to react with the sulfonium intermediate to form the sulfenic acid on Cys72 and release Met as a product (VI). After the release of Met, the Cys72 sulfenic acid form (VII) can be reduced back to the thiolate (I) by reducing agents, including the C-terminal resolving cysteines or DTT. The sulfenic acid can also undergo reversible dehydration to form a sulfenamide by reaction with either a side chain amino group or an amide group in the backbone, a reaction favored in the electrospray mass spectrometer. The left side of the figure shows the proposed mechanism for the MsrA oxidase. Met binds to the sulfenic acid form of MsrA in the same orientation as MetO does in the reductase cycle and with water bound to Glu115 and Tyr103 (VIII). A lone pair of electrons on the Met sulfur attacks the sulfenic sulfur in a SN2 (bimolecular nucleophilic substitution) manner to form the positively charged sulfonium intermediate (IX) and release a molecule of water. Another molecule of water, presumably again bound in the pocket with Glu115 and Tyr103, attacks the sulfonium sulfur to form the sulfurane intermediate (X). Glu115 then acts as a general base to abstract a proton from the hydroxyl group of the sulfurane intermediate. The sulfur–sulfur bond breaks to form MetO and Cys72 is regenerated (XI). With release of MetO, the reduced form of MsrA is formed (I).

Materials and Methods

Nonmyristoylated wild-type and mutant MsrA were expressed and purified as described (39). For each preparation the mass was checked by HPLC-mass spectrometry and was always within 0.5 Da of the calculated mass. The methodology was as described except that the mass spectrometer was an Agilent model 6200, which was also utilized for peptide mapping and sequencing by tandem mass spectrometry (40). To determine the stoichiometry of Met formation, 1 μM MsrA was incubated with different concentrations of l-methionine sulfoxide (MetO) without reducing agents in 50 mM sodium phosphate pH 7.5 with 1 mM diethylenetriaminepentaacetic acid (DTPA). The reaction was stopped by adding 10% trifluoroacetic acid to a final concentration of 0.5%. Met was determined by amino acid analysis (41). S-MetO and R-MetO were separated on an APEX column after derivatization (1). For detection of sulfenic acid, 1 μM MsrA was incubated with 0.5 mM 5, 5-dimethyl-1, 3-cyclohexanedione (dimedone) or NBD-chloride (NBD) for 1 h at 37 °C. To determine susceptibility to oxidation, peptides and proteins were incubated with the C107S/C218S/C227S/Δ228–233 MsrA in the presence of 10 mM MetO for 6 h at 37 °C. This mutant lacks the last six residues at the C terminus and the three Cys other than the active site Cys72 were mutated to Ser. Kinetic parameters were determined with wild-type MsrA using the hexapeptide as substrate with Met or MetO (American Peptide), varying the concentrations from 0.25 to 10 mM. The initial rate at different substrate concentrations was determined after 5 (reductase) or 15 min (oxidase) incubation at 37° from the area of the hexapeptide on the HPLC reverse phase chromatogram at 210 nm and fit to Michaelis–Menton kinetics.