Previous observations have suggested a link between oxidative stress and a growth requirement for methionine in E. coli. We investigated the duration of growth lags induced by oxidative stress in cells growing in glucose-minimal medium containing or lacking methionine. In the experiment shown in Figure 2A and 2B, oxidative stress was induced by addition of the thiol oxidant diamide, which rapidly penetrates cells and oxidizes intracellular thiols. Diamide treatment does not kill cells, but results in an abrupt arrest of growth for a length of time that is dependent on the initial concentration of diamide. Cells resume normal growth once the thiol status is restored (Kosower and Kosower 1995). E. coli cultures logarithmically growing in glucose-minimal medium with and without methionine experienced a lag in growth upon challenge with diamide (Figure 2A). However, cells grown in medium containing methionine resumed growth significantly faster than those needing to synthesize methionine. Higher diamide concentrations increased the duration of the growth lag and augmented the effect of methionine on resumption of growth.

The biosynthesis of methionine involves several reactions (see Figure 1). If a step upstream of the reaction catalyzed by MetE were responsible for methionine limitation during diamide treatment, addition of homocysteine to the medium should decrease the growth lag imposed by diamide. No effect of homocysteine addition was seen on the duration of the growth lag in diamide-treated cells (Figure 2B), even though homocysteine can support the growth of cells that lack enzymes upstream of MetE in the biosynthetic pathway (Urbanowski and Stauffer 1989).

Similar results were obtained when hydrogen peroxide (H₂O₂) was used to induce oxidative stress. Addition of methionine to the medium significantly decreased the growth lag produced by H₂O₂ treatment (Figure 2C), while homocysteine was ineffective and even amplified the lag (Figure 2D). The mechanism by which homocysteine appears to have potentiated the H₂O₂ stress is not known; however, it was clearly not protective. Taken together, the growth experiments suggest that methionine becomes limiting under oxidative stress, and that the bottleneck to de novo biosynthesis of methionine lies in the terminal step catalyzed by MetE.

The methionine auxotrophy observed under oxidative stress conditions could reflect an increased demand for methionine or a decreased rate of de novo synthesis due to inactivation of MetE. To distinguish between these possibilities, we assayed the activity of the purified enzyme upon challenge with GSSG (Figure 3). Nearly complete loss of methionine synthase activity was observed following addition of GSSG. This process was readily reversible upon incubation with a reductant such as dithiothreitol (DTT) or reduced glutathione (GSH) (data not shown). Activity loss was correlated with thiol oxidation, as assessed by dithio-1,4-nitrobenzoic acid (DTNB) titration of the trichloroacetic acid (TCA)–precipitated protein. GSSG treatment led to thiol oxidation, which proceeded with approximately the same rate constant (0.06 min⁻¹) as loss of activity (0.07 min⁻¹).

While the rate of thiol oxidation could be measured with DTNB, the absolute number of cysteines oxidized was ambiguous. MetE contains seven cysteines, yet DTNB titration of the fully reduced enzyme gave values between 7.2 and 9.0 cysteines for different protein preparations, and oxidation of MetE led to an average decrease of 1.8 cysteines per mol. Thus it was important to use independent means to determine the number of cysteines oxidized by GSSG.

Oxidation of MetE by GSSG would be expected to proceed by initial formation of a mixed disulfide between MetE and glutathione (equation 2).

An inter- or intramolecular disulfide bond could then be generated by attack on the mixed disulfide by a second cysteine residue. These two possibilities can be distinguished by using mass spectrometry to determine the molecular mass of the oxidized protein. The reduced protein was found to be 84,530 ± 6 Da (expected mass, 84,542, −0.01% deviation), whereas the mass of the protein oxidized by GSSG was 84,835 ± 8 Da. The mass difference of 305 ± 10 Da between oxidized and reduced MetE is consistent with the addition of a single glutathione adduct (expected mass, 306 Da). Formation of an intramolecular disulfide bond would only decrease the mass by 2 Da, which would not be detected by mass spectrometry in such a large protein.

To obtain evidence for stoichiometric glutathionylation of MetE, the oxidized protein was reduced with DTT to release the glutathione adduct. The DTT and GSH were separated from the protein using a Microcon concentrator. The filtrate was assayed for GSH using fluorescamine, which reacts with primary amines to form a fluorescent product. The reduced protein did not release GSH (0.0 mol GSH per mol protein) while oxidized protein released 0.9 mol GSH per mol of protein. Thus both mass spectrometry and the fluorescamine assay were consistent with glutathionylation of MetE by GSSG.

In order to determine the site of oxidation and verify that an intramolecular disulfide was not being formed concomitant with oxidation, we performed disulfide mapping experiments based on the method of Wu and Watson (1997). MetE was cyanylated with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under denaturing conditions at pH 3, and then cleaved immediately N-terminally to the cyanylated cysteines in 1 N ammonia, resulting in the formation of 2-iminothiazolidine-4-carboxyl (itz) peptides (Figure 4). Cleavage will not occur at cysteines that have been protected from cyanylation by oxidation. Following cleavage, any disulfide bonds were reduced with DTT and the resulting peptide fingerprint analyzed by liquid chromatography mass spectrometry (LC-MS).

This approach allows for relatively straightforward mapping of disulfide bonds. The acidic pH employed minimizes the possibility of disulfide bond scrambling. Furthermore, the ability to cleave N-terminally to each cyanylated cysteine allows cleavage between closely spaced, even adjacent, cysteines. These factors provide a significant advantage over traditional methods of peptide mapping, particularly for MetE, which contains cysteines spaced only two residues apart at the active site (cysteines 643 and 645). Moreover, for large proteins, most proteolytic and chemical cleavage methods have the potential to generate numerous peptides, whereas this approach produces a more limited number.

LC-MS analysis of the cyanylated and cleaved reduced MetE allowed for identification of all but one of the expected peptides (Figure 5A; Table 1). High performance liquid chromatography (HPLC) peaks with masses corresponding to each of the expected peptides were observed, except for the dipeptide itz643–644, which is too small to be detected. Cyanylated cysteines can also undergo a β-elimination side reaction instead of cleavage. Susceptibility of cyanylated cysteines to β-elimination depends on the neighboring residues (Wu and Watson 1998). In MetE, cysteine 726 readily formed the β-elimination product I, and β-elimination at cysteine 560 produced a small amount of product H. Identification of these side products provides additional evidence that cysteines 560 and 726 were reduced and available for cyanylation. Disulfide mapping of a mutant MetE, MetEC>A1–4, in which cysteines 323, 353, 516, and 560 were changed to alanines, further confirmed the mass assignments. Only peaks corresponding to cleavage next to the three remaining cysteines (643, 645, and 726) were observed (Figure 5B; Table 1).

Disulfide mapping data for the oxidized protein following cyanylation, cleavage, and reduction with DTT (Figure 6A; Table 1), were similar to results for the reduced protein, suggesting that most of the cysteines in MetE remained reduced. Peptides A, B, C, D, E, and H provide evidence that cysteines 323, 353, 516, 560, and 643 were reduced. Peptides G and J indicate that cysteine 726 was reduced. The pattern is consistent with the initial oxidation occurring at cysteine 645 alone. Peptide J is the expected product obtained when MetE is initially oxidized at cysteine 645, with β-elimination at cysteine 726, and is then reduced.

LC-MS analysis of the oxidized protein fragments prior to DTT reduction yielded an almost identical pattern to that obtained after reduction (Figure 6B; Table 1). However, a peptide, K, corresponding to itz643–753 with a glutathione adduct and β-elimination, was observed instead of J. It is not possible to distinguish which cysteine, 645 or 726, is glutathionylated and which has undergone β-elimination based simply on the masses of peptides J and K. However, the presence of peptide G in all traces at a level similar to that of reduced MetE indicates that cysteine 726 must be reduced. Moreover, the high susceptibility of cysteine 726 to undergo β-elimination (as evidenced by peak I in Figure 5A)—contrasted with the complete lack of a β-elimination product at cysteine 645—argues that β-elimination occurs at cysteine 726 in the oxidized enzyme and glutathionylation occurs at cysteine 645.

In order to confirm that cysteine 645 is the only site of oxidation, the inactivation of various MetE mutants was analyzed. MetEC>A1–4 contains only cysteine 645 and the zinc-binding ligands, cysteines 643 and 726; the latter ligands are required for catalytic activity. This mutant has approximately 90% of the activity of the native protein, and the kinetics of inactivation by GSSG are nearly identical to that of the wild-type protein (Figure 7A). However, mutation of cysteine 645 to alanine (MetEC>A5) dramatically altered the course of inactivation by GSSG. The slow partial loss of activity seen with MetEC>A5 may reflect weakening of zinc binding for the mutant enzyme, allowing glutathione to compete with MetE for zinc.

Further evidence implicating cysteine 645 as the site of sensitivity to oxidation was obtained by monitoring the oxidation of MetE by diamide. The reaction of diamide with thiols can be followed at 325 nm, where diamide absorbs strongly, but the reduced product does not (Kosower and Kosower 1995). Addition of MetE to diamide results in a rapid decrease in absorbance at 325 nm (Figure 7B). However, MetEC>A5 does not appear to be oxidized by diamide. This observation suggests that diamide oxidation of MetE in vitro involves an initial attack of cysteine 645 on diamide to form an inactive covalent complex (Figure 7C). In the presence of accessible thiols, such as the millimolar concentrations of GSH present in vivo, this complex can then readily react to form glutathionylated MetE and reduced diamide. Thus, although diamide oxidation does not result in glutathionylation of MetE in the absence of glutathione, this experiment demonstrates the extreme reactivity of cysteine 645 to oxidizing agents.

In order to further characterize the effects of GSSG oxidation on MetE, the reduced and oxidized forms of the protein were subjected to gel filtration. The elution of glutathionylated MetE was markedly shifted in comparison to that for the reduced protein (Figure 8A). The change in hydrodynamic radius seen upon oxidation suggested that GSSG oxidation had altered the conformation of MetE. Therefore, to confirm a conformational difference between reduced and oxidized MetE, we analyzed the two forms of the protein by circular dichroism (CD) spectroscopy and limited tryptic digestion. The similarity between the CD spectra of oxidized and reduced MetE suggests that the oxidized protein has not undergone a gross structural rearrangement and is still largely intact (Figure 8B). However, the deviation in the two spectra between 210 and 230 nm is consistent with a conformational change upon glutathionylation. Tryptic digestion of oxidized MetE appeared to occur with different kinetics compared to that of the reduced protein (Figure 8C). The oxidized protein was more stable against tryptic digestion than the reduced form, again consistent with a conformational change upon MetE oxidation.

MetE has previously been shown to be an in vivo substrate of the chaperone GroEL (Houry et al. 1999). Since oxidation of MetE was found to induce a conformational change, we tested whether GroEL bound preferentially to either form of MetE. GroEL and reduced MetE were incubated together and then injected onto a gel filtration column (Figure 8D). Separate peaks corresponding to GroEL and reduced MetE were observed, indicating that reduced MetE did not associate with GroEL. However, when the glutathionylated protein was incubated with GroEL, only the GroEL peak was observed. The GroEL complex is large enough (approximately 800 kDa) that binding of MetE would not be expected to shift the elution time significantly. Thus, the disappearance of the peak corresponding to oxidized MetE strongly suggests that GroEL specifically binds glutathionylated MetE, leading to coelution.

To gain a better understanding of the thermodynamic sensitivity of MetE to oxidation, we measured the equilibrium constant for formation of the inactive mixed disulfide. MetE was incubated with varied ratios of GSH and GSSG until equilibrium was reached, and then activity was assayed (Figure 9A). The equilibrium constant K_mix (equation 2) was determined to be 1.4 and found to be independent of GSH concentration. If formation of an intramolecular disulfide bond were responsible for MetE inactivation, the equilibrium constant should be dependent on the GSH concentration (Gilbert 1995). The independence of the equilibrium constant from GSH concentration provides further support that glutathionylation is the mechanism of inactivation.

In order to estimate the extent that MetE inactivation may be expected in vivo, both the redox potential, which is determined by the [GSH]²/[GSSG] ratio, and the total glutathione concentration in the cell ([GSH] + 2[GSSG]) need to be taken into account. Based on the equilibrium constant for GSSG oxidation, MetE activity at equilibrium was calculated for typical cellular glutathione concentrations (1–5 mM [Kosower and Kosower 1978; Åslund et al. 1999]) at various potentials as determined by the GSH–GSSG redox couple (Figure 9B). (At each potential, E_h, the ratio of [GSH]²/[GSSG] was calculated from the Nernst equation:

where E_o is –0.252 V at pH 7.2 (using a value of –0.24 V at pH 7.0 with an adjustment of –6.2 mV per 0.1 increase in pH [Schafer and Buettner 2001]), R is 8.314 V·C/mol·K, T is 310 K, n is 2, and F is 96,485 C/mol. Based on a total glutathione concentration ([GSH] + 2[GSSG]) of 1, 2, or 5 mM, and the calculated [GSH]²/[GSSG], [GSH] and [GSSG] can be determined at each cellular potential. The ratio of [GSH]/[GSSG] may then be used to calculate the relative amount of MetE activity expected at equilibrium by

where K_mix is 1.4.)

For E. coli growing under normal conditions (–250 mV to –280 mV [Hwang et al. 1992]), more than 97% of the cellular MetE would be expected to be reduced and active. However, in a severely oxidizing environment (greater than –150 mV), inactive MetE may accumulate to an appreciable level. In comparison, Hsp33, which is a redox-regulated chaperone, has a midpoint potential of –170 mV (Jakob et al. 1999). Therefore, thermodynamically, under conditions where Hsp33 is 50% active, MetE would be expected to be 29%–54% oxidized for glutathione concentrations of 1–5 mM. Although GSSG activates Hsp33 via formation of an intramolecular disulfide bond and inactivates MetE by glutathionylation, this suggests that both proteins may have similar sensitivities to oxidation.

However, these calculations assume that the oxidation state of MetE is in equilibrium with GSH and GSSG. Deviations from theoretical values could occur if MetE were oxidized or reduced in vivo by a mechanism other than simple thiol-disulfide exchange. Comparison of the actual amount of oxidized MetE to measurements of the physiological concentrations of GSH and GSSG could therefore provide an indication of the in vivo mechanism of oxidation. Nevertheless, while MetE inactivation by GSSG may be energetically favorable under oxidizing conditions, the apparently slow kinetics make it difficult to conclude whether or not oxidation actually occurs in vivo.

Thus, to determine whether our observations are physiologically relevant, we needed to develop a method whereby the oxidation of MetE could be directly observed in vivo. To this end, thiol-trapping experiments were performed as outlined in Figure 10A. Cells growing under normal or oxidative conditions were briefly incubated with iodoacetamide, which is able to quickly penetrate the cell membrane and efficiently alkylate all available reactive thiols. The proteins were precipitated with TCA, and oxidized cysteines were reduced with DTT. The sulfhydryls exposed under these conditions were trapped with iodoacetic acid, which adds a negative charge to the protein for each cysteine trapped. Differently charged forms of the protein were separated by vertical slab isoelectric focusing, and MetE was visualized by immunoblotting. This method of analysis should readily distinguish between glutathionylated MetE (one additional negative charge as compared to reduced MetE) and MetE with an intramolecular disulfide bond (two additional negative charges as compared to reduced MetE). Glutathionylated MetE prepared in vitro is more acidic than fully reduced MetE, as indicated by comparing lanes 4 and 3 in Figure 10B.

In vivo thiol-trapping experiments were performed with logarithmically growing wild-type and trxA gor strains of E. coli. Strains mutant in genes for one of the thioredoxins (trxA) and glutathione reductase (gor) are impaired in their ability to reduce disulfide bonds, resulting in a highly oxidizing cytoplasmic environment (Prinz et al. 1997). Comparison of the thiol status of MetE in a trxA gor mutant strain (WP843) with that in the isogenic wild-type strain (DHB4) revealed that approximately 50%–60% of the MetE in the trxA gor strain was oxidized in vivo (Figure 10B). Moreover, the shift in the isoelectric point for MetE that was oxidized in vivo is identical to the mobility of glutathionylated MetE prepared in vitro.

E. coli strains containing mutations in trxB (which specifies thioredoxin reductase) and gor experience more severe disulfide stress than trxA gor strains (Prinz et al. 1997). Cells require the presence of DTT in the medium to grow at rates comparable to wild-type strains; growth rapidly halts when DTT is removed. This strain cannot be grown in minimal medium, where wild-type MetE is expressed, so a trxB gor strain containing a plasmid specifying MetE (which expresses MetE independently of the cellular methionine concentration) was grown in Luria-Bertani (LB) medium containing DTT. Oxidized MetE was observed within 90 min of DTT removal, while MetE was fully reduced in the presence of DTT (Figure 11A and 11B). Again, the shift in the isoelectric point of MetE oxidized in vivo was indistinguishable from that of glutathionylated MetE prepared in vitro. Together, these experiments provide evidence that MetE is glutathionylated during steady-state growth in two different strains of E. coli that are intrinsically oxidatively stressed.

In order to establish a direct link between oxidative inactivation of MetE and the methionine auxotrophy observed during transient oxidative stresses, we performed thiol-trapping experiments on cells challenged with diamide. When wild-type E. coli cells growing exponentially in glucose-minimal medium were treated with 0.9 mM diamide, MetE was found to be completely oxidized during the lag in growth (Figure 11C and 11D). Identical levels of reduced MetE reappeared within 60 min of the challenge, just prior to the resumption of normal cellular growth. Thus oxidation of MetE is associated with the cellular methionine limitation (described earlier) that is imposed by addition of diamide. Furthermore, these results are consistent with those of Leichert and Jakob (2004), which independently show that MetE is rapidly oxidized in E. coli (wild-type strain DHB4) within 2 min of diamide treatment.

Electrophoresis-grade urea, Chaps, ampholytes (pH 4–8), ammonium persulfate, TEMED, 10 N sodium hydroxide, and 85% phosophoric acid were obtained from Genomic Solutions (Ann Arbor, Michigan, United States). Iodoacetate and iodoacetamide were purchased from Fluka (Sigma-Aldrich, St. Louis, Missouri, United States). (6S)-5-methyltetrahydropteroyltriglutamate was synthesized from pteroyltriglutamate (Schircks Laboratories, Jona, Switzerland), as described previously (Matthews 1986). L-Homocysteine was prepared by hydrolysis of L-homocysteine thiolactone (Drummond et al. 1995). Polyclonal rabbit antibodies to purified wild-type MetE were generated by Lampire Biological Laboratories (Pipersville, Pennsylvania, United States). All other chemicals were obtained from Sigma (St. Louis, Missouri, United States). GroEL was generously provided by Z. Xu (University of Michigan, Ann Arbor, Michigan, United States).

Wild-type K-12 strain W3110 was obtained from F. C. Neidhardt (University of Michigan, Ann Arbor, Michigan, United States). Wild-type E. coli K-12 strain DHB4 and isogenic E. coli K-12 strains WP843 (trxA gor) and WP778 (trxB gor) (Prinz et al. 1997) were obtained from U. Jakob (University of Michigan). Overexpression of mutant MetE proteins was performed in E. coli K-12 strain GW2531 (metE) (Mulligan et al. 1982). The MetE mutants MetEC>A5 and MetEC>A1–4 were constructed by overlap extension PCR (Ge and Rudolph 1997) using Pfu Turbo (Strategene, La Jolla, California, United States). Plasmid pJG816 (González et al. 1992), specifying wild-type MetE, was used as the template, and the primers used for overlap extension are shown in Table 2. The products were purified by gel electrophoresis and ligated into alkaline phosphatase–treated pJG816. Plasmids containing the mutant genes were isolated after transformation into competent XL1-blue cells by electroporation. Single Cys→Ala mutations of Cys323 (pEM1), Cys353 (pEM2), Cys560 (pEM4), and Cys645 (pEM5) were constructed by overlap extension using pJG816 as the template. For the construction of pEM3 (Cys353Ala, Cys516Ala), pEM2 was used as the template instead of pJG816. For construction of pEM1, pEM2, pEM3, and pEM4, the purified products and pJG816 were digested with BssHII and StuI, while for pEM5, AatII and BssHII were used. MetEC>A1–4 was constructed by subcloning of fragments containing the individual mutations from the appropriate plasmids. Plasmids pEM1 and pEM3 were digested with MluI; the fragment containing the Cys323Ala mutation from pEM1 was ligated into the alkaline phosphatase–treated pEM3 vector to generate pEM6. Similarly, pEM6 was digested with AgeI and BssHII and then ligated into pEM4 that had been digested with the same enzymes to generate pEM7. Plasmids containing pEM5 (MetEC>A5) and pEM7 (MetEC>A1–4) were transformed into E. coli strain GW2531. Mutations were confirmed by DNA sequencing of the entire metE gene.

Wild-type MetE was expressed using strain GW2531/pJG816, MetEC>A5 was expressed using strain GW2531/pEM5, and MetEC>A1–4 was expressed from strain GW2531/pEM7. Six 1-l portions of LB medium supplemented with 100 μg/ml ampicillin and 0.5 mM zinc sulfate were inoculated with 3 ml of an exponentially growing culture in the same medium (OD₆₀₀ ∼1). Cultures were incubated at 37 °C with shaking at 250 rpm for approximately 30 h before harvesting. The proteins were purified as described previously (González et al. 1992), except that an 800-ml gradient from 100 to 400 mM potassium phosphate (pH 7.2) was used. Purified proteins were dialyzed into 50 mM potassium phosphate buffer (pH 7.2) containing 500 μM DTT, concentrated, and stored at –80 °C.

For examining the effect of oxidative stress in minimal medium, cultures were grown aerobically at 37 °C in a rotary water bath shaker in glucose-minimal morpholinopropane sulfonate (MOPS) medium (Neidhardt et al. 1974) supplemented with 10 μM thiamine. Exponentially growing cultures in the same medium were diluted to an OD₆₀₀ of approximately 0.02–0.04. Where diamide was added to cultures, the concentration of the diamide stock was determined spectrophotometrically (ɛ₂₉₆ = 3,000 M⁻¹cm⁻¹) (Kosower and Kosower 1995). H₂O₂ concentrations were determined similarly (ɛ₂₄₀ = 43.6 M⁻¹cm⁻¹) (Hildebrandt and Roots 1975; Poosch and Yamazaki 1986).

The molar absorption coefficient for MetE at 280 nm was determined to be 157,000 ± 1000 M⁻¹cm⁻¹ using the Endelhoch method (Pace et al. 1995). The specific activity of MetE was measured using the modified enzyme assay previously described (González et al. 1996), but omitting DTT from the assay mix. Exclusion of DTT did not affect the measured activity. For experiments monitoring enzyme inactivation, aliquots containing 650 pmol of MetE were withdrawn from the oxidation reaction and mixed with activity assay reagents which had been pre-equilibrated to 37 °C. The final assay mixture contained 10 mM potassium phosphate buffer (pH 7.2), 50 mM Tris chloride (pH 7.2), 100 μM magnesium sulfate, 2 mM L-homocysteine, 66 μM (6S)-5-methyltetrahydropteroyltriglutamate, and enzyme in a total volume of 400 μl. After a 30-s incubation at 37 °C, the reaction was quenched by addition of 100 μl of 5 N hydrochloric acid/60% formic acid, followed by heating at 88 °C for 10 min. After cooling on ice, the reaction was centrifuged at 14,000g for 5 min at 4 °C to remove precipitated protein. The amount of tetrahydrofolate product (converted to methenyltetrahydrofolate by formic acid) was quantitated by its absorbance at 350 nm. Excess GSSG used to oxidize the enzyme did not significantly alter activity during the 30-s assay.

MetE (50 μM) in 100 mM Tris chloride (pH 7.2) was equilibrated to 37 °C, and GSSG was added (final concentration, 5 mM) to initiate the oxidation reaction. The GSSG concentration was established by assay with glutathione reductase (Zander et al. 1998). At time points, aliquots were removed for measurement of cysteine oxidation or enzyme activity (described above). To determine the extent of oxidation, aliquots (100 μl) of the reaction mixture were removed and quenched with 100 μl of ice-cold 20% TCA (w/v). Samples were incubated on ice at least 30 min and then pelleted by centrifugation at 14,000g for 30 min at 4 °C. The supernatant was removed, and the pellet was dissolved in 100 mM potassium phosphate buffer (pH 7.3) containing 0.25 mM DTNB, 6 M guanidinium chloride, and 1 mM EDTA. Complete dissolution of the pellet required vortexing for 15–20 min. Reduced cysteines were quantitated by measuring the absorbance at 412 nm after subtraction of a blank containing DTNB and buffer but no protein. The extent of oxidation was determined using the fraction of reduced cysteines at the indicated time compared to the value at time zero (taken as 100%).

Purified MetE was desalted into 100 mM Tris chloride (pH 7.2) to remove DTT from the storage buffer. The reduced protein (50 μM) was then incubated with 10 mM GSSG for 90 min at 37 °C. Samples were concentrated using Microcon concentrators (Millipore, Billerica, Massachusetts, United States), and GSSG was removed using Bio-Gel P-6 columns (BioRad, Hercules, California, United States) equilibrated with 100 mM Tris chloride (pH 7.2). Reduction and reactivation of GSSG-oxidized MetE was accomplished by incubation of the oxidized enzyme with 2 mM reductant at 37 °C. For preparation of samples of diamide-oxidized MetE to be used as standards for in vivo thiol-trapping assays, diamide (1 mM) was incubated with MetE (10 μM) in 100 mM Tris chloride (pH 7.2) at 37 °C. After 45 min, diamide was removed by gel filtration using Bio-Gel P-6 columns.

Reduced and GSSG-oxidized MetE preparations were exchanged into 50 mM potassium phosphate (pH 7.2) using Bio-Gel P-6 columns. Proteins were then washed three times with the same buffer using Centricon concentrators. MetE (50 μM) was incubated with 2 mM DTT in 50 mM potassium phosphate (pH 7.2) in a volume of 200 μl for 1 h at 37 °C. Each solution was then loaded into a Microcon concentrator and centrifuged at 12,000g for 5 min at room temperature. Aliquots (100 μl) of the filtrate were added to 1.7 ml of 200 mM borate buffer (pH 9.0) containing 1 mM p-(hydroxymercuri)benzoic acid. While vortexing, 0.6 ml of 1 mg/ml fluorescamine (in acetone) was added. The fluorescence emission of the samples was measured at 485 nm with excitation at 390 nm. A range of GSH concentrations (0–125 mM) was assayed to generate a standard curve. GSH concentrations were determined by DTNB titration (Riddles et al. 1983). The stoichiometry of glutathionylation was calculated by subtracting the results obtained in the absence of DTT from the amount of glutathione released by incubation with DTT and dividing by the enzyme concentration (50 μM). Each form of MetE was assayed in duplicate from two separate preparations.

CDAP dissolved in 200 μl of 100 mM citrate buffer (pH 3) containing 6 M guanidine hydrochloride was added to 100 μl of MetE in 100 mM Tris chloride (pH 7.2) and incubated for 15–20 min at room temperature. The stock concentrations were adjusted so that CDAP was approximately 25-fold in excess over the total thiol concentration. CDAP was removed from the solution by gel filtration, using Bio-Gel P-6 columns equilibrated with 6 M guanidine hydrochloride solution (brought to pH ∼7–8 with potassium hydroxide). Concentrated ammonium hydroxide was added (final concentration 1 M [pH ∼12]), and the reaction was incubated at room temperature for approximately 75 min. Samples were then lyophilized to remove the ammonia and stored at –80 °C. Prior to LC-MS analysis, DTT was added (final concentration was approximately 200 times the concentration of MetE) and allowed to incubate at room temperature for 30 min. Solutions were then diluted into 6 M guanidine hydrochloride solution (adjusted to pH 3 with acetic acid) and characterized by LC-MS.

Samples (250–500 pmol) were injected onto a Vydac (Hesperia, California, United States) 250 mm × 2.1 mm C4 column (214MS52) using an analytical HPLC Surveyor from ThermoFinnigan (San Jose, California, United States). The column was equilibrated with 97% solvent A (water with 0.1% v/v acetic acid and 0.02% v/v TFA) and 3% solvent B (acetonitrile with 0.1% v/v acetic acid and 0.02% v/v TFA), and samples were eluted at a flow rate of 0.25 ml/min with a three-step linear gradient: 3% B for 15 min, 3% to 30% B in 9 min, and 30% to 65% B in 35 min. For LC-MS of the holo-protein, samples were desalted using a two-step linear gradient: 20% B for 10 min, and 20% to 70% B in 25 min. Fractionation was monitored by the UV chromatogram recorded at 280 nm. The eluent was diverted to waste for the first 15 min, and then directly infused into the electrospray ionization source of a ThermoFinnigan LCQ mass spectrometer. Mass spectral data were obtained in the positive mode, and ESI spectra were deconvoluted using the BioMass software provided by ThermoFinnigan.

Oxidation of MetE by diamide was monitored at 37 °C using a Hi-Tech Scientific (Salisbury, United Kingdom) SF-61DX2 stopped-flow spectrophotometer equipped with a xenon light source for single wavelength detection. Diamide (1 mM) and MetE (100 μM) in 100 mM Tris chloride (pH 7.2) were mixed, and reduction of diamide was monitored at 325 nm.

MetE (20 pmol) was injected onto an Alltech (Lexington, Kentucky, United States) macrosphere size exclusion HPLC column (250 mm × 4.6 mm) with a 300-Å pore size (7-μm particle size) and run at 0.5 ml/min with 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride. For GroEL interaction experiments, GroEL (2 μM) was incubated with MetE (1 μM) in 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride for 30 min at 37 °C before analysis by size exclusion chromatography.

Reduced and oxidized MetE were buffer exchanged into 10 mM potassium phosphate buffer (plus 500 μM DTT for the reduced protein) (pH 7.2). The final filtrates were collected and used as a blank. Samples were centrifuged for 1 h at 14,000g at 4 °C and then diluted to 0.4 mg/ml. Far UV CD spectra were recorded using a JASCO (Easton, Maryland, United States) J-810 spectropolarimeter and 0.1 cm cuvettes. Absorbance spectra of the same sample were recorded on a JASCO V-550 UV/Vis spectrophotometer to determine the precise protein concentration. The data were then normalized to a protein concentration of 4.5 μM MetE.

Aliquots containing 2.5 mg/ml reduced or oxidized MetE in 50 mM potassium phosphate (pH 7.2) were digested with 0.02% (w/v) trypsin at room temperature. At indicated times, 5-μl samples were quenched with 10 μg of 1-tosylamido-1-lysyl chloromethylketone, and then analyzed by SDS-PAGE (10% gel).

MetE (10 μM) was incubated with varying concentrations of GSH and GSSG in 100 mM Tris chloride (pH 7.2) at 37 °C. GSH (Riddles et al. 1983) and GSSG (Zander et al. 1998) stock concentrations were measured as described. After 5.5 h, aliquots were removed and MetE activity was assayed. Since A₃₅₀ directly correlates to the amount of product in the activity assay, relative MetE activity was calculated by:

where A₃₅₀(x) is the absorbance at 350 nm for sample x, A₃₅₀(oxid) is for completely oxidized MetE (2 mM GSSG, no GSH), and A₃₅₀(red) is for completely reduced MetE (2 mM GSH, 4 μM [trace] GSSG). The equilibrium constant, K_mix, was then determined by plotting relative MetE activity versus the [GSH]/[GSSG] ratio.

All E. coli cultures used for in vivo thiol-trapping experiments were grown aerobically at 37 °C. E. coli wild-type (DHB4) and trxA gor (WP843) strains were grown in glucose-minimal MOPS medium supplemented with 10 μM thiamine and all amino acids except methionine (Neidhardt et al. 1974), with 0.4 mM arginine used instead of 5.2 mM. Tetracyclin (20 μg/ml) was included in the medium for the trxA gor strain. Overnight cultures of exponentially growing cells were diluted to an OD₆₀₀ of approximately 0.02 into the same medium. Cultures were allowed to grow to an OD₆₀₀ of approximately 0.4, and samples were removed for thiol trapping as described below.

For in vivo thiol-trapping experiments performed on the trxB gor strain, a plasmid expressing MetE (pJG816) was transformed into electrocompetent trxB gor (strain WP778) cells by electroporation. An overnight culture of WP778/pJG816 in LB medium containing 20 μg/ml kanamycin, 50 μg/ml ampicillin, and 4 mM DTT was diluted to an OD₆₀₀ of approximately 0.02 into the same medium (without kanamycin). Care was taken to ensure that an overnight culture from the same inoculum failed to grow when DTT was not included in the medium. The 50-ml culture was grown to an OD₆₀₀ of approximately 0.4, and then filtered into a prewarmed sterile filter flask. The cells were washed twice with prewarmed sterile LB medium to remove residual DTT, and resuspended in 50 ml of prewarmed LB medium. At the indicated time points following DTT removal, samples were taken for alkylation as described.

To assay the oxidation status of MetE in diamide-treated cells, wild-type E. coli were grown in glucose-minimal MOPS medium without methionine as in the growth experiments described above, except that diamide (0.9 mM, final concentration) was added at an OD₆₀₀ of approximately 0.4. Aliquots were removed at intervals following diamide addition for treatment with alkylating agents.

In order to trap oxidized thiols in vivo, 1 ml of culture was added to 250 μl of 0.5 M iodoacetamide in 100 mM Tris chloride (pH 7.2) that had been pre-equilibrated at 37 °C. Samples were incubated 2 min at 37 °C, quenched by the addition of 140 μl of 100% (w/v) ice-cold TCA, and incubated on ice for at least 30 min. Precipitated proteins were pelleted by centrifugation (14,000g) for 30 min at 4 °C, washed with cold 10% TCA (once) followed by ethanol (twice), and then dried by incubating approximately 20 min in a 37-°C incubator. The pellet could then be stored at –80 °C prior to secondary alkylation with iodoacetic acid. Pellets were dissolved in 50 μl of alkylation buffer containing DTT (0.1 M Tris chloride [pH 8.7] containing 0.2 M potassium chloride, 1 mM EDTA, 8 M urea, and 10 mM DTT), and incubated 15 min at room temperature. Newly exposed cysteines were trapped by adding 12.5 μl of 0.5 M iodoacetate (104 mg dissolved in 0.5 ml of 1 M potassium hydroxide, followed by addition of 0.5 ml of 1 M Tris chloride [pH 7.3]) and incubating 2 min at room temperature. For some samples, excess DTT was added to quench the reaction; however, this was found to not be necessary. Alkylated proteins were then frozen in liquid nitrogen and stored at –80 °C prior to analysis by isoelectric focusing.

For the reduced and oxidized MetE standards, 25 μl of 0.5 M iodoacetamide was added to 100 μl of 10 μM MetE in 100 mM Tris chloride (pH 7.2) and incubated for 1 min at 37 °C. The reaction was quenched by addition of 125 μl of 20% (w/v) ice-cold TCA and incubation on ice for at least 30 min. The proteins were pelleted by centrifugation (14,000g) at 4 °C for 30 min and then washed with cold 10% TCA (once) and either ether (3 times) or 1 M Tris chloride (pH 7.2) followed by water. Pellets were then alkylated with iodoacetate as described for the in vivo culture samples.

The vertical slab isoelectric focusing protocol developed was based on that of Savinova and Jagus (1997) and Robertson et al. (1987). Gels contained 8 M urea, 9.6% glycerol, 3.75% acrylamide, 5% ampholytes, and 1.25% Chaps. Briefly, 6.0 g of urea was dissolved in 2.4 ml of 18 MΩ water, 2.4 ml of 50% glycerol, and 1.56 ml of acrylamide solution (28.36% acrylamide/1.62% bis-acrylamide). 600 μl of ampholytes (pH 4–8) and 780 μl of 20% Chaps were added, and the resulting solution was degassed for approximately 10 min. 25 μl of 10% APS and 20 μl of TEMED were added, and 8 cm × 8 cm × 1.0 cm gels were poured. Gels were allowed to polymerize approximately 30 min and then used immediately. Lanes were carefully washed with cathode buffer.

The sample buffer solution contained 9.5 M urea, 5% chaps, 4.5% glycerol, 0.5 mM lysine–hydrochloride, 2% ampholytes (pH 4–8), 50 mM DTT, and 0.15% SDS. Care was taken not to increase the solution temperature above 30 °C to avoid breaking down urea, which could then carbamylate proteins. Aliquots of the sample buffer were snap-frozen in liquid nitrogen and stored at –20 °C.

An Xcell Surelock Mini-cell gel apparatus from Invitrogen was assembled with the freshly prepared gel, and degassed cathode (upper, 0.1 M sodium hydroxide) and anode (lower, 10 mM phosphoric acid) solutions were added. Samples were diluted at least 10-fold into sample buffer and loaded. Power was applied for 1 h at 100 V followed by 1 h at 200 V and finally 30 min at 500 V. Gels were then stained as described previously (Garfin 1990) or blotted.

Gels were electroblotted onto PVDF membrane (Amersham Pharmacia, Piscataway, New Jersey, United States) according to the manufacturer's (Invitrogen) protocol using 1/2X Towbin buffer. Proteins were immunoblotted with a polyclonal rabbit antibody to MetE (1:3,000) and immune complexes were revealed using a fluorescein-linked anti-rabbit antibody (ECF western blotting kit, Amersham Pharmacia) followed by direct detection using a Molecular Dynamics (Sunnyvale, California, United States) Storm phosphorimager.

The Swiss-Prot (http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are AhpC (P26427), glutaredoxin 1 (P00277), glutaredoxin 2 (P39811), glutaredoxin 3 (P37687), glutathione reductase (P06715), GroEL (P06139), iron superoxide dismutases (P09157), manganese superoxide dismutase (P00448), MetA (P07623), MetE (C. albicans) (P82610), MetE (E. coli) (P25665), MetE (T. maritima) (Q9X112), MetH (P13009), OxyR (P11721), PAPS reductase (P17854), thioredoxin 1 (P00274), thioredoxin reductase (P09625), and Tpx (P37901).