A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

Abstract

B₁₂-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.

Methionine synthase (MetH) catalyses the transfer of a methyl group to homocysteine (Hcy) to form methionine, with its cobalamin cofactor serving as an intermediary. During catalysis the methylcobalamin cofactor is demethylated by Hcy to form cob(I)alamin and methionine and then remethylated by methyltetrahydrofolate (CH₃H₄folate) to form tetrahydrofolate (H₄folate) and regenerate methylcobalamin (MeCbl). The MetH catalytic cycle is shown in red in Fig. 1. Under microaerophilic conditions, cob(I)alamin is oxidized to the catalytically inactive cob(II)alamin form approximately once in every 2,000 turnovers (1). To return Escherichia coli MetH to the catalytically competent methylcobalamin form, the cofactor is reduced by flavodoxin and methylated by S-adenosyl-l-methionine (AdoMet) (2, 3), a reactivation sequence shown in blue in Fig. 1. MetH can thus access two alternative methyl donors, CH₃-H₄folate and AdoMet, as well as one methyl acceptor, Hcy. To prevent futile cycling of cob(I)alamin during the catalytic cycle, the enzyme discriminates against AdoMet; during the reactivation sequence, the enzyme similarly discriminates against CH₃H₄folate (4). These studies have shown that the cob(I) alamin form of the enzyme is unable to switch between the conformations necessary for catalysis and reactivation. However, the structural basis for such discrimination remained unknown.

Fig. 1.

The catalytic (red) and reactivation (blue) cycles of E. coli MetH. During catalysis the cobalamin cofactor is alternately methylated by CH₃-H₄folate and demethylated by Hcy. The cob(I)alamin form of the enzyme is occasionally oxidized to form the inactive cob(II)alamin form. Return of this species to the catalytic cycle involves reduction with electrons derived from reduced flavodoxin and methylation with a methyl group derived from AdoMet.

MetH is a modular enzyme (Fig. 2) with four functional units that bind Hcy, CH₃-H₄folate, cobalamin, and AdoMet (5). The crystal structures of the N-terminal modules of MetH from Thermotoga maritima and the C-terminal modules from E.coli have been solved (6–8). The Hcy- and folate-binding modules are (βα)₈ barrels, with the sites for methylation of Hcy and demethylation of CH₃-H₄folate separated by ≈50 Å (6). The structure of the isolated cobalamin-binding module (residues 651–896) revealed that the cobalamin is sandwiched between two domains: a four-helix bundle (Cap domain) lying over the upper (β) face of the cobalamin and a Rossman α/β domain (Cob domain) that provides binding determinants for the lower (α) face of the cobalamin (7). This conformation is referred to as Cap:Cob in Fig. 2. Nε2 of His-759 in the Cob domain is coordinated to cobalt in the α position. Nδ1 of His-759 is also hydrogen bonded to Asp-757, which in turn is hydrogen bonded to Ser-810. This set of residues, known as the ligand triad, helps secure the histidine ligand and plays an important role in determining the distribution of conformers during catalysis (9, 10). When His-759 is coordinated to the cobalt, the enzyme is said to be in the His-on state, with the MeCbl cofactor exhibiting an absorbance maxima at 525 nm (red). His-off MeCbl has an absorbance maximum at 450 nm (yellow) that can readily be distinguished from the His-on form, which in principle allows quantitation of the fraction of enzyme in the reactivation conformation (9).

Fig. 2.

Cartoon of a minimal set of conformations for the methylcobalamin forms of MetH(2–1227) (Upper) and MetH(649–1227) (Lower). The Hcy-binding module is shown in green, the folate-binding module in light yellow, the cobalamin-binding domain in red, the Cap domain in dark yellow, and the AdoMet-binding domain in blue. The corrin ring of the cobalamin is represented by the square, and the vertical line below it indicates the His-on conformation of the Hcy:Cob, Fol:Cob, and Cap:Cob conformations. In the AdoMet:Cob conformation, the histidine is displaced and the corrin moves away from the cobalamin-binding domain to assume a His-off conformation.

A structure of a C-terminal fragment comprising the Cob and AdoMet modules of catalytically inactive His759Gly MetH(649–1227) in the cob(II)alamin form has also been determined (8), demonstrating a dramatic rearrangement of the Cap and Cob domains that allows access of the AdoMet-binding module to the lower (β) face of the cobalamin. This conformation is referred to as AdoMet:Cob in Fig. 2. The cap is translated 25 Å from the position it occupies in the structure of the isolated cobalamin domain and the corrin moiety moves away from the Cob domain toward the AdoMet binding module. The cobalt is 2.3 Å further away from C_α of Gly-759 in the AdoMet:Cob structure than in the Cap:Cob structure of the isolated cobalamin domain. This structural rearrangement, if physiologically relevant, will be accompanied in the WT protein, whether full-length or the C-terminal fragment, by bond dissociation between His-759 and cobalt, thus leading to the formation of a His-off cob(II)alamin species.

The aforementioned molecular rearrangement is only one of the several rearrangements that MetH undergoes: each of the methyl transfer reactions shown in Fig. 1 occurs at a different substrate-binding domain and requires a different arrangement of modules (Fig. 2). During the catalytic cycle, the enzyme oscillates between a conformation with the CH₃-H₄folate-binding and Cob domains positioned for methylation of cobalamin (Fol:Cob) and a conformation with the Hcy and Cob domains positioned for demethylation (Hcy:Cob). Recent studies have examined the distribution of molecular species in the MeCbl form of MetH and shown that the enzyme exists as an ensemble of interconverting conformations. The distribution of states at equilibrium depends on the oxidation and ligation state of the cobalamin, on the temperature, and on the concentrations of substrates or products (9, 11).

In this study, we have used disulfide cross-linking to lock the C-terminal fragment of WT MetH into the reactivation conformation before crystallization. Specifically, we engineered unique cysteine residues positioned to form a disulfide that would span the Cap and Cob domains of MetH. The I690C/G743C MetH(649–1227) becomes locked in the reactivation conformation. The axial ligand to the cobalamin moiety, His-759, is dissociated from the cobalamin. Relative to the distance from C_α of His-759 to cobalt in the Cap:Cob conformation (7), the cobalt is displaced by 2.9 Å in the reactivation conformation. Unexpectedly, the displaced His-759 is now involved in intermodular contacts with the AdoMet module. The Nε2 nitrogen of His-759 interacts with the AdoMet domain directly through Asp-1093 and via a water molecule to Glu-1069. These interactions would be expected to stabilize the AdoMet:Cob conformation, and may rationalize the failure of the cob(I)alamin form of MetH to interconvert between AdoMet:Cob and Fol:Cob conformations of the enzyme.

Results and Discussion

Introduction of a Disulfide Cross-Link to Reduce the Conformational Flexibility of MetH.

A remaining challenge for understanding the complex reaction sequence of MetH is to understand how the domains interact and rearrange during the reaction cycle. Crystal structures of the full length enzyme in the different conformations are essential for achieving this goal. Unfortunately, crystallization of the full-length enzyme has been unsuccessful to date, presumably due to the conformational flexibility of MetH. A different strategy was thus required to tackle this problem, and we chose originally to implement it in the C-terminal fragment of MetH. We used an intramodular disulfide cross-link to reduce the conformational flexibility of MetH.

The structure of the C-terminal MetH fragment in the AdoMet:Cob conformation was used to select residues suitable for mutation to cysteines (8). The stereochemical criteria for selecting appropriate residues are described in ref. 12. The residues chosen were in different domains of the cobalamin module, with Ile-690 in the Cap domain and Gly-743 in the linker connecting the Cap and Cob domains. This disulfide cross-link is expected to tether the enzyme in the AdoMet:Cob conformation because it shortens the nine residue linker region between the Cap and Cob domains by four residues and is positioned so as to restrict movement of the Cap relative to the Cob domain. To eliminate the possibility of intramolecular disulfide linkages between the cysteine mutants and the native cysteines at positions 772, 1042 and 1142 of MetH(649–1227), the two cysteine mutations had to be introduced at positions with C_α separations of at least 6 Å or more from each of the native cysteines.

Expression and Characterization of I690C/G743C MetH(649–1227).

The mutant enzyme was overexpressed and purified to >95% purity as judged by electrophoretic analysis under denaturing conditions and is isolated as a mixture of cob(II)alamin and aquacobalamin forms. Reductive methylation in an electrochemical cell in the presence of AdoMet converted the protein to the methylcobalamin form. The methylated enzyme is very light sensitive, so to prevent photolysis of the methyl-cobalt bond, all subsequent experiments were performed in the dark. Whereas WT MetH(649–1227) at room temperature gave a spectrum consistent with His-on MeCbl, with an absorbance maximum at 525 nm (ε₅₂₅ = 9,400 M⁻¹ cm⁻¹), the spectrum of I690C/G743C MetH(649–1227) showed a maximum at 452 nm and an ε₄₅₂ of 10200 ± 100 M⁻¹ cm⁻¹. This blue shift of the λ_max compared with WT MetH indicates that I690C/G743C MetH(649–1227) is mostly His-off.

I690C/G743C MetH(649–1227) Is Found in both His-On and -Off States.

The methylcobalamin form of both WT and I690C/G743C MetH(649–1227) showed temperature-dependent changes in absorbance consistent with an interconversion between a His-off species at high temperature and a His-on species at low temperatures, as shown in Fig. 3. These changes were used to calculate equilibrium constants [K = (% His-off)/(% His-on)]. From the plot of ln K vs. T⁻¹, we calculated ΔH = 27.5 kcal/mol and ΔS = 83.2 cal/mol/K for the interconversion between His-on and His-off species of WT MetH(649–1227). These values yielded a ΔG of + 1.73 kcal/mol at 37°C, where the positive value indicates a preference to remain in the His-on conformation. A value for ΔG of + 2.55 kcal/mol was determined previously for the full-length WT enzyme (11). This more positive value is probably due to the larger number of His-on conformations in the full-length enzyme (Fig. 2). Similar experiments were performed with the I690C/G743C double mutant. ΔH was calculated to be 6.2 kcal/mol and ΔS = 26.9 cal/mol/K, yielding a ΔG of −2.1 kcal/mol at 37°C. The negative value of ΔG seen for the mutant reflects the preference for the enzyme to be in the His-off form, presumably attributable to the stabilizing effect of the disulfide cross-link on this species.

Fig. 3.

The effect of temperature on the absorbance of WT MetH(649–1227) (A and B) and I690C/G743C MetH(649–1227) (C and D). The spectra were recorded in 10 mM potassium phosphate buffer, pH 7.2, after equilibration for 2 min at 10°C (purple), 15°C (deep blue), 20°C (light blue), 25°C (green), 30°C (light green), and 37°C (orange). Arrows indicate the direction of absorbance changes as the temperature is increased. The equilibrium constant, K = (%His-off/%His-on), was calculated at each temperature to create van't Hoff plots, shown on the right. Thermodynamic parameters were calculated as described in Methods.

In contrast, the aquacobalamin form of I690C/G743C MetH(649–1227) is red at all temperatures, consistent with the very high affinity of Co³⁺ in aquacobalamin for a coordinated base in the lower axial position (13). Surprisingly, the cross-linked mutant structure can assume a His-on conformation (see below).

Thiol Quantitation.

The free thiols were quantitated by assaying with DTNB. WT MetH(649–1227) enzyme contains a total of three free thiols (Table 1), as expected from the amino acid sequence. The I690C/G743C enzyme contains a total of five cysteines, but only three free thiols were found by DTNB titration, indicating the presence of a disulfide cross-link.

Table 1.

Sulfhydryl group quantitation by DTNB titration

Enzyme	Cysteines based on		Disulfides
	Sequence	DTNB assay
WT MetH(649–1227)
Methylcobalamin form	3	2.9 ± 0.1	0
Aquacobalamin form	3	3.0 ± 0.2	0
I690C/G743C MetH(649–1227)
Methylcobalamin form	5	2.7 ± 0.1	1
Aquacobalamin form	5	2.9 ± 0.1	1

The titrations were performed at room temperature as described in Methods.

To verify that the intramodular disulfide involved the introduced cysteines, we performed disulfide mapping experiments based on the method of Wu et al. (14) and Hondorp et al. (15). The data are shown in supporting information (SI) Table 2 and SI Fig. 7. The five cysteines of the mutant protein are located at residues 690, 743, 772, 1042, and 1142, and of these, 690 and 743 are predicted to form a disulfide bond. Mass spectrometric analysis of the cyanylated and cleaved I690C/G743C MetH(649–1227) allowed the identification of all but one of the expected peptides. Repeated attempts by mass spectrometry proved unsuccessful in identifying peptide 1–772, which would contain the disulfide cross-link, although peptides indicating cleavage at cysteines 772, 1042, and 1142 were observed. The absence of the disulfide cross-link between Cys-690 and Cys-743 would have given rise to two extra peptide fragments containing residues 1–689 and 690–771, but no evidence was found for the presence of these peptide fragments. Thus, the evidence from DTNB titrations and mass spectrometry suggests the presence of a disulfide bond between cysteines 690 and 743.

Overall Structure of I690C/G743C MetH(649–1227).

We obtained crystals of the mutant MetH at 37°C, where the enzyme was mostly in the His-off conformation. The overall architecture of I690C/G743C MetH(649–1227) is shown in Fig. 4. Crystallographic information as well as refinement statistics are provided in SI Table 3. The enzyme was crystallized in the activation conformation where the helical cap region and the cobalamin binding module are adjoined to each other with the AdoMet binding module on top of the cobalamin moiety. Cys-690 is positioned at the beginning of helix Ia3 in the cap domain (7) and Cys-743 is in the linker region connecting the Cap and Cob domains and is located in a flexible region of the protein. This flexibility is reflected in the poor electron density corresponding to this loop in both the His759Gly mutant structure previously determined (8) and in the present disulfide cross-linked structure. Consequently, we cannot unequivocally model the two cysteine side chains in the observed electron density. However, whereas the position of the side chains of the residues in that area cannot be well defined, the backbone is clearly defined and the C_αα distance of 5.4 Å is consistent with a disulfide. The electron density in this region is shown in SI Fig. 8. Moreover, as predicted, the distances between the native and the engineered cysteines are long enough to preclude disulfide bond formation. Thus, although the presence of the disulfide bond cannot be unambiguously demonstrated by crystallography, the evidence based on DTNB titrations, mass spectrometry, thermodynamic parameters, and the overall conformation of the enzyme in the structure, is consistent with the presence of a Cys-690–Cys-743 disulfide cross-link.

Fig. 4.

A ribbon drawing of I690C/G743C MetH(649–1227). The activation domain (blue) caps the corrin ring and interacts with the cobalamin-binding domain (red) and the four-helix bundle or cap (yellow). The cysteine residues are shown in space filling rendition, and the cobalamin cofactor (CPK coloring) is rendered in stick.

The structure of I690C/G743C MetH(649–1227) is very similar to that of the previously determined His759Gly MetH(649–1227) (a comparison of the two structures is shown in SI Fig. 9). When the two structures are compared, each of the individual domains is very similar, and they move as rigid bodies relative to one another. The cobalamin-binding domain in the disulfide cross-linked structure is now closer to the AdoMet module and the cofactor, although the position of the corrin ring relative to the AdoMet module is basically unchanged. As will be discussed below, the closer approximation of the AdoMet and Cob domains may be a result of the new intermodular contacts made by His-759.

The Cobalamin has Photolyzed.

No electron density was observed at either face of the cobalamin moiety near the cobalt atom, indicating the absence of the methyl group in the crystal structure and a resulting four-coordinate cobalt species (Fig. 5). SI Fig. 10 shows the electron density. The result was unexpected because the crystallization trays were set up with the MeCbl form of I690C/G743C MetH(649–1227). Although care was taken to expose the enzyme and the crystal to as little light as possible, the I690C/G743C MetH(649–1227) is more susceptible to photolysis than the WT enzyme. The demethylation was possibly due to photoreduction during preparation of the crystals and/or photoreduction due to exposure to high-energy x-ray to form cob(II)alamin, as previously observed in other determinations of the structures of cobalamin-containing enzymes (16–19). Magnetic circular dichroism studies of the cob(II)alamin formed by photolysis of methylated I690C/G743C MetH(649–1227) in a rigid glass at 77 K has established that the cobalamin is four-coordinate (unpublished data obtained in collaboration with Matthew Liptak and Thomas Brunold, University of Wisconsin, Madison).

Fig. 5.

A comparison of the active site of I690C/G743C MetH(649–1227) in the His-off conformation (Right) and the cobalamin binding module of WT MetH (651–896) (Left), in the His-on conformation. The cobalamin cofactor undergoes significant movement relative to its position in the isolated cobalamin binding module; note in particular the tilting of the equatorial plane of the corrin ring in the structure of I690C/G743C MetH(649–1227), which moves the cobalt away from C_α of His-759 by 2.9 Å. The side chain of His-759 undergoes a dramatic reorientation, whereas the positions of the distal residues comprising the catalytic triad, Asp-757, and Ser-810 are relatively unchanged between the two conformations.

Intermodular Constraints in the His-Off Conformation.

In the previously reported structure of H759G MetH(649–1227) in the reactivation conformation (AdoMet:Cob), the critical His-759 residue was absent, so the position of this important ligand, when not bound to Co, and its potential role to the enzyme while in a “His-off state” was hitherto unknown. The presence of this ligand in our structure provides us with the opportunity to assess the differences as well as the similarities between the His-off and His-on states where the latter is represented by the structure of WT MetH (651–896) (7). As shown in Fig. 5, a very substantial rearrangement of His-759 in the His-off conformation is evident when compared with the structure of the isolated cobalamin fragment in the His-on Cap:Cob conformation. In the His-off state His-759 not only moves away with respect to Co as evident by the C_α displacement of 2.9 Å, but the histidine ring also rotates and faces away from the Co atom. However the differences between the His-on and His-off states, based on these two structures, appear to be only in the His-759 and cobalamin positions. Although the cobalamin and histidine ring seem to be moving away from each other in going from the His-on to the His-off form, other residues in the cobalamin domain, including the other two residues of the catalytic triad (Ser-810 and Asp-757), align well with each other.

Although His-759 is not bound to cobalt, it still interacts with cobalamin through a hydrogen bond between Nδ1 and one of the propionamide side chains. More importantly, His-759 makes specific contacts with the AdoMet module. In I690C/G743C MetH(649–1227) His-759 forms a hydrogen bond with Asp-1093 and a water mediated hydrogen bond to Glu-1069 (Fig. 6). These two residues are both located in the AdoMet-binding module of MetH. Asp-1093 is a highly conserved residue among all MetH homologues, whereas the consensus sequence for MetH indicates a polar residue at the position of Glu-1069. Reduction of His-on cob(II)alamin to His-off cob(I)alamin in WT MetH occurs with uptake of a proton, presumably on His-759 (20). The interactions shown here are consistent with protonation of His759Nε2, thus identifying Asp-1093 as a potential proton donor. Thus, this interaction may result in the formation of a hydrogen bond between members of an ion pair. Such His⁺–Asp⁻ hydrogen bonds may contribute as much as 3–5 kcal/mol of stability (21).

Fig. 6.

Intermodular interactions involving His-759 in the His-off state. Nε2 of His-759 interacts with the AdoMet module directly through a hydrogen bond to Asp-1093 and via a water-mediated hydrogen bond to Glu-1069. Nδ1 of His-759 forms a hydrogen bond with the amide of the propionamide side chain of ring B of the cobalamin (data not shown).

This intermodular interaction is an unanticipated finding and could provide a structural basis for the inability of the cob(I) alamin form of MetH to interconvert between catalytic and reactivation conformations. When the His-759 and Gly-759 forms of the reactivation conformation are compared, a major difference is the intermodular bonding between His-759 and Asp-1093, and the water-mediated interaction between His-759 and Glu-1069. These intermodular interactions should significantly stabilize the His-759 enzyme in the reactivation conformation and prevent interconversion to the catalytic conformation while the cobalamin is in a His-off state [e.g., cob(I)alamin].

A low-resolution (3 Å) structure of the aquacobalamin form of I690C/G743C MetH(649–1227), which is always in the red His-on state, confirms that the enzyme is indeed in the AdoMet:Cob conformation (data not shown). In this structure, the histidine has rotated back toward the cobalt of the cobalamin and is now close enough to serve as a ligand and the Cob domain has moved closer to the cobalamin. After methylation by AdoMet, the enzyme might transiently convert to the His-on species in this conformation, weakening the intermodular interactions and allowing the enzyme to convert to a catalytic conformation. It will now be of great interest to ascertain whether similar intermodular interactions between His-759 and the Fol module in the His-off cob(I)alamin formed by demethylation during catalytic turnover also serves to lock the enzyme in the Fol:Cob conformation, preventing access of the cobalamin to AdoMet and ensuring its access to CH₃-H₄folate.

Conclusions

In summary, we solved a structure of a C-terminal fragment of methionine synthase that was stabilized by disulfide cross-linking. The cross-linking significantly reduced the conformational flexibility of this fragment, favoring the His-off reactivation conformation by ≈3.9 kcal/mole. The His-off state of His-759 in the reactivation conformation is stabilized by its intermodular interactions with the AdoMet binding module. The movement required to dissociate the histidine is limited to the side chain of this residue, with the other catalytically important residues in the cobalamin-binding domain being unaffected.

Methods

Expression and Purification of MetH(649–1227) and I690C/G743C MetH(649–1227).

Amino-terminal His₆-tagged E.coli WT MetH(649–1227) was generated by Vahe Bandarian (University of Arizona, Tucson, AZ) from pMMA11 and pT7-H plasmids by using restriction digestion and ligation. The resultant plasmid pVB8 enables the expression of His-tagged C-terminal MetH. The enzyme was expressed in cells of E.coli Hms174(DE3) (Novagen) containing the pVB8 plasmid. Bacteria were grown at 37°C and harvested as described in ref. 22. Cells were resuspended to a density of 0.5–0.75 g of cells per ml in 20 mM sodium phosphate (pH 7.2), with N^α-p-tosyl-l-lysine chloromethyl ketone (10 μg/ml) and phenylmethylsulfonyl fluoride (50 μg/ml). Cells were then lysed by sonication, and unbroken cells were removed by ultracentrifugation for 45 min at 40,000 × g.

Sodium chloride (500 mM)/20 mM sodium phosphate (pH 7.3) was added to the lysate, and 10–15 ml of the lysate was applied to a HiTrap chelating column (Amersham Biocsciences, Uppsala, Sweden) charged with nickel sulfate and was equilibrated with 500 mM sodium chloride/20 mM sodium phosphate (pH 7.3). The column was washed with four column volumes of this buffer and four volumes of 20 mM imidazole/500 mM sodium chloride/20 mM sodium phosphate (pH 7.3). MetH(649–1227) was eluted with 200 mM imidazole/500 mM sodium chloride/20 mM sodium phosphate (pH 7.3). The enzyme was then exchanged into 10 mM potassium phosphate (pH 7.2) and loaded on a Mono Q 16/10 column (Amersham Biosciences) equilibrated with the same buffer. The protein was eluted with a 300-ml linear gradient between 10 and 400 mM potassium phosphate buffer. The colored fractions were pooled together, concentrated in an Amicon concentrator and exchanged into 10 mM potassium phosphate buffer, pH 7.2. Protein concentration was calculated by using the absorbance of the bound MeCbl cofactor (ε₅₂₄ = 9400 M⁻¹ cm⁻¹). The yields ranged from 7 to 9 mg of purified MetH(649–1227) per liter of cell culture.

The I690C/G743C double mutant was introduced into the pVB8 vector using the QuikChange site-directed mutagenesis kit (Stratagene). The primers were obtained from Invitrogen. The sequence of I690C/G743C MetH(649–1227) was confirmed by complete sequencing at the Biomedical Research Core Facility of the University of Michigan. I690C/G743C MetH(649–1227) was purified as described above for the WT fragment. Protein concentration was calculated by using the absorbance of the bound His-off MeCbl cofactor (ε₄₅₂ = 10200 M⁻¹ cm⁻¹) after the protein was reductively methylated in the presence of AdoMet (23).

Reduction, Methylation, and Photolysis of Cobalamin.

I690C/G743C MetH(649–1227) was converted to the MeCbl form by reductive methylation in an electrochemical cell using AdoMet as the methyl donor (23). The cob(II)alamin form of MetH was formed by the photolysis of MeCbl MetH as described in ref. 23.

Crystallization and Cryoprotection.

The 65-kDa I690C/G743C MetH(649–1227) fragment in 50 mM Tris buffer at pH 7.2 was concentrated to ≈12 mg/ml. Crystals were grown by slow evaporation in microbatch plates under oil (3:1 mixture of paraffin and silicon oil) by mixing 2 μl of protein with 2 μl of a solution that consists of 0.2 M potassium nitrate and 20% (wt/vol) PEG3350. The crystallization trays were kept at 37°C for 1 week before they were transferred to 22°C, where they were kept until crystals were harvested. Crystals were transferred to a solution of 18% (wt/vol) PEG3350, 0.2 M potassium nitrate, and 15% propylene glycol in 25 mM Tris buffer at pH 7.0 for a few minutes before they were flash frozen.

Data Collection and Structure Determination.

Diffraction data were collected at GM/CA-CAT (Advanced Photon Source, Argonne National Laboratory, Argonne, IL) on a Mar300 detector and processed with XDS (24). The crystal structure of H759G MetH(649–1227) from E. coli (PDB ID 1K7Y) was used as a search model after the removal of the cobalamin cofactor, water molecules, and residues from flexible regions with high B factors. Phases were obtained by molecular replacement with the program EPMR (25). Initial structure refinements with CNS included simulated annealing in torsional space, coordinate minimization and restrained individual B-factor adjustment with maximum-likelihood targets (26). No sigma(F) data cutoff was used in refinement. A disulfide restraint between Cys-690 and Cys-743 was introduced toward the latter stages of refinement. A final restrained refinement using isotropic B factors with REFMAC5 was implemented (27). Model modification was performed with XtalView/Xfit (28). The geometric quality of the model and its agreement with the structure factors was assessed with PROCHECK and SFCHECK, respectively (29, 30). Figures were generated by using Pymol (31).

Determination of Cobalamin Extinction Coefficients and Concentrations.

The extinction coefficients of the enzyme-bound cofactor species of the MetH(649–1227) and I690C/G743C MetH(649–1227) were determined as described for the MeCbl-bound state of full-length WT MetH (32).

Determination of Thermodynamic Parameters Associated with the His-On/His-Off Conversion.

MetH(649–1227) (4–6 μM) in 50 mM potassium phosphate buffer (pH 7.2) was placed in a sample cuvette in a dual beam Varian Cary Bio 300 UV-Vis thermostated spectrophotometer and an equal amount of buffer was added to the reference cuvette. The samples were allowed to equilibrate for 2 min at the desired temperature before spectra were recorded. Spectral deconvolution was used to determine the fractions of His-on and His-off species in the observed spectrum as described by Bandarian et al. (9). The thermodynamics of the conversion of His-off to His-on forms of MetH was determined by initially calculating the equilibrium constant (K = % His-off/% His-on) at each temperature, and then using those values to construct a van't Hoff plot. The enthalpy (ΔH) and entropy changes (ΔS) were calculated from a linear fit to Eq. 1

The free energy differences were calculated from Eq. 2

Similar experiments were performed with I690C/G743C MetH(649–1227).

Thiol Titration with 5,5′-Dithio-bis(2-nitrobenzoic Acid) (DTNB).

The free thiol content of MetH(649–1227) and I690C/G743C MetH(649–1227) samples was analyzed by reaction with DTNB using an ε₄₁₂ of 13600 M⁻¹ cm⁻¹ (33). Enzyme solutions were prepared either under native conditions in 0.05 M Tris·HCl (pH 8.0)/1 mM EDTA or after denaturation. After reaction with DTNB, the absorbance at 412 nm was measured at 25°C.

Mapping the Disulfide Bond by Liquid Chromatography and Mass Spectrometry.

The protocol for mapping the position of the disulfide bond in samples by LC-MS is included in SI Text.