The heart of the mer HgR mechanism is mercuric ion reductase (MerA), an enzyme that catalyzes the conversion of the thiol-avid Hg(II) to volatile, uncharged Hg(0) that lacks significant affinity for any liganding functional groups. The enzyme utilizes NADPH as source of electrons  and is located in the cytoplasm  where this substrate is plentiful. However, thiols of proteins and smaller molecules that are the primary target for tight binding by Hg(II) are also plentiful in this location. Consequently, the efficiency of the reductase at competing with these cellular thiols to scavenge and reduce the incoming metal ion is critical to the survival of the cell, and significant research has focused on understanding the features of the protein that are essential for this process.
Although MerA was purified in the early 1970s from both Pseudomonas K62 and E. coli[191–194], significant mechanistic studies [195–198] were not initiated until after the discovery of a FAD cofactor  in the enzyme. Fox and Walsh  reported the first large-scale purification of the Tn501-encoded MerA and showed that the homodimeric enzyme exhibited spectral and biochemical properties similar to those of the pyridine nucleotide disulfide oxidoreductase family whose most common members include glutathione reductase (GR) and lipoamide dehydrogenase. Partial peptide sequences  confirmed the relationship by the presence of a highly homologous active site region with the two cysteines that form the characteristic redox-active disulfide/dithiol of the family. However, unlike the other family members that use this cysteine pair for catalysis and are inhibited when Hg(II) binds to them , MerA has additional structural features to help it avoid such inhibition.
Two unique regions, each with a pair of cysteines, were identified in the Tn501 merA gene: a short C-terminal extension, and a lengthy N-terminal extension of ca. 95 amino acids . On the basis of the X-ray structure of human GR , and the MerA/GR homology, it was predicted that the C-terminal cysteines of one monomer would lie near the redox-active cysteines of the other monomer and could assist in Hg(II) binding at the active site , a prediction proven true by a wide variety of biochemical and structural studies described below. On the other hand, the lack of homology in the N-terminal extension suggested that this region would form a separate domain with its two cysteines involved in acquiring Hg(II) and handing it off to the cysteines in the catalytic core . Early studies provided no evidence for a function of the N-terminal extension, and further analysis of this domain was delayed until very recently. The latter will be discussed in Section 3.4.2.
Elucidation of the structural organization and roles of the four core cysteines in the catalytic mechanism has been a major focus of research on the protein. As noted above, the redox-active cysteines in other disulfide reductases cycle between the disulfide (Eox) and dithiol (EH2) states in each round of catalysis. Pyridine nucleotide substrates for those enzymes reduce the disulfide in Eox to the dithiol in EH2 but need not be bound to EH2 during the reduction of the disulfide substrate by the enzyme dithiol . Although an analogous mechanism was considered a possibility for MerA , chemical precedent for the interaction of thiols with Hg(II) suggested a role for high-affinity complexation of Hg(II) by these thiols rather than a role in its reduction. The first evidence in favor of the simple binding role came from the observation that Hg(II) could bind to the inner (i.e. redox-active) cysteines of EH2 in MerA but was not reduced unless another molecule of NADPH was also bound . Consistent with this, it was found that the EH2 form of MerA binds NADPH very tightly [197,203,204], such that in the cell the enzyme would be primed with its binding site and bound reductant waiting for Hg(II) to enter the cell. Although these results suggested that the inner cysteines participate only in Hg(II) binding, the catalytic competence of the EH2–Hg complex (in terms of rate of reduction) was not tested and left open the possibility of a mechanism involving binding of Hg(II) to only one of the inner cysteines in EH2–NADPH followed by oxidation of the pair concomitant with reduction of Hg(II) and then rapid reduction of the disulfide by the bound NADPH [205,206].
Further distinction between these possibilities as well as analysis of the roles of the C-terminal cysteines in the process has relied heavily on studies using enzymes with single, double or triple site-directed mutations of the four cysteines. Mutation of the redox-active cysteines to either serine  or alanine [208,209] confirmed an orientation of these cysteines relative to FAD identical to that found in GR, as well as an essential, but still ambiguous role for the pair in catalysis.
Mutation of the C-terminal cysteines to alanines provided several key observations in the elucidation of the roles of all four cysteines. The double mutant (CCAA, cysteines listed in order of primary sequence) showed no activity in steady-state assays even though it could form the EH2–NADPH complex and bind Hg(II) [210,211]. This suggested that the Hg(II) complex of the inner cysteines alone was not a reducible complex . At the same time, careful reductive titrations of the wild-type protein demonstrated that most preparations required four electrons to reach a stable EH2 state (instead of the expected two electrons) and that each two electrons gave rise to the appearance of two new cysteine thiols in the protein for a total of four  rather than the two previously reported }. The CCAA mutant however took only two electrons to form the stable EH2 indicating that the extra disulfide in the ‘hyperoxidized’ wild-type enzyme was at the C-terminus . Consistent with this, the hyperoxidized wild-type enzyme showed no initial activity when enzyme was added last to steady-state assays . However, it was fully active after a short preincubation with NADPH [212,213] suggesting that the C-terminal disulfide was in sufficient proximity to the inner cysteines to undergo reduction via disulfide interchange with them. This was corroborated by the observation that reaction of the hyperoxidized Eox enzyme with one equivalent of NADPH led first to rapid reduction of the inner cysteines followed by reoxidation concomitant with the appearance of two new cysteine thiols . The combination of these results confirmed the original proposal by Brown et al.  that the C-terminal cysteines lie near the active site and play some role in binding Hg(II).
In a clever subsequent study, equal amounts of the inactive double mutant of the inner cysteines (AACC) and the inactive double mutant of the C-terminal cysteines (CCAA) were mixed under conditions allowing dissociation, scrambling and reassociation of the monomers into dimers. The resulting mix exhibited 25% of the wild-type activity as expected for a statistical mix of 25% of each inactive homodimer and 50% heterodimers with only one of two active sites reconstituted with four cysteines , a result consistent with the structural prediction that the C-terminal cysteines from one monomer form an active site with the inner cysteines of the other monomer in the homodimeric enzyme . Final confirmation of these structural predictions appeared in the X-ray crystal structure of MerA from Bacillus sp. RC607, a model of which is shown in Fig. 3.
Figure 3. Model of MerA from crystal structure of Bacillus enzyme . Functional groups of conserved residues in binding pathway and active site are shown in spacefill model: yellow-Cys sulfur, red-Tyr hydroxyl, blue-Lys amino.
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Single CysAla mutations of the C-terminal cysteines were used to further probe their individual roles. In vivo evaluation of either single or the double CysAla mutants demonstrated an essential role for both for in vivo resistance [210,215]. In spite of this, only the double mutant showed no activity in vitro . While one of the single mutants was more impaired, the other exhibited ca. 50% of the wild-type kcat. However, both had significantly lower kcat/KM values than wild-type indicating that both C-terminal cysteines are important in the acquisition of Hg(II) from solution and suggesting that a lowered efficiency at scavenging Hg(II) was the major cause of lost resistance for these two mutants . The retention of activity in the single mutants but complete lack of any steady-state activity in the CCAA mutant, in spite of its ability to bind Hg(II), also suggested that one of the C-terminal cysteines might be involved in formation of the actual reducible Hg(II) complex along with one or both of the inner cysteines [212,215]. A two-coordinate complex involving one C-terminal and one inner cysteine would be consistent with the mechanism involving reduction of Hg(II) via initial oxidation of the inner cysteines [205,206], while a three-coordinate complex would be consistent with the simple binding role for the inner cysteines.
Some support for the mechanism involving the two-coordinate complex came from the crystal structure of a Cd(II) complex of the Bacillus MerA . Cd(II), which has too low a potential to be reduced but inhibits the enzyme, was found to bind in a distorted tetrahedron involving one inner and one C-terminal cysteine and two conserved tyrosines in the active site . As the C-terminal cysteines are clearly involved in the acquisition of Hg(II) in vivo, such a complex for Hg(II) would certainly be expected to occur at some point in the binding process. However, the observation of this binding site for Cd(II) in MerA may simply reflect both its higher affinity for oxygen ligands and greater preference for tetrahedral coordination than found for Hg(II) .
The critical evidence that finally established a simple binding role for the inner cysteines and ruled out any participation of the C-terminal cysteines in formation of the reducible Hg(II) complex came from pre-steady-state kinetic studies of the wild-type and CCAA enzymes [217–219]. In these studies, three HgX2 complexes with ligands of varying size and basicity were examined as substrates in the reaction with the NADPH complex of EH2, the competent species for reduction of Hg(II) . The most important finding was that with HgBr2 as substrate, the CCAA enzyme bound and reduced Hg(II) as rapidly as the wild-type enzyme in both single and multiple turnovers [217,219]. This result alone demonstrated that neither C-terminal cysteine is required to form the reducible Hg(II) complex and, together with the spectral changes during the course of the reaction, ruled out the mechanism involving reduction of Hg(II) via initial oxidation of the inner cysteines. The data clearly indicate that Hg(II) forms a complex with the two inner cysteines and then is reduced by electrons transferred through the flavin from the bound NADPH [217,219].
The reason for the discrepancy between this surprising rapid pre-steady-state turnover by the CCAA enzyme versus the non-existent steady-state activity of this mutant became clearer when the pre-steady-state reactions were examined with Hg(CN)2 and Hg(Cys)2, the latter one being the substrate in the steady-state assays. Although these two substrates reacted with drastically different rate constants (see below), they formed spectrally identical Hg(II) complexes with the enzyme in which the bound Hg(II) was not reduced . The spectrum of this non-reducible Hg(II) complex differs from that of the reducible complex formed using HgBr2 as substrate in a way that suggests buildup of negative charge in the active site during displacement of the more basic CN− and thiolate ligands from the respective HgX2 substrates. The presence of a negative charge in the vicinity of the bound Hg(II) would be expected to inhibit the input of electrons needed to reduce it. These results suggested then that the C-terminal cysteines provide an essential ligand exchange pathway that removes the basic thiolate ligands before Hg(II) reaches the inner active site thereby avoiding buildup of negative charge at the site of reduction .
As expected, the wild-type enzyme received and reduced Hg(II) from the Hg(Cys)2 substrate at a catalytically competent rate in the pre-steady-state reaction. However, the reaction with Hg(CN)2 varied with the number of equivalents used in the reaction. With excess Hg(CN)2 the same inhibited complex was formed as observed in the CCAA enzyme, while with one equivalent, about half of the Hg(II) was reduced at a catalytically competent rate. These results suggested that Hg(CN)2 can access the inner cysteines of the wild-type enzyme via two pathways with different outcomes. Direct reaction with the inner cysteines, as must occur in the CCAA enzyme, leads to the inhibited complex, while reaction via the C-terminal ligand exchange pathway, as must occur with Hg(Cys)2 in the wild-type enzyme, leads to the reducible Hg(II) complex . This result provided further evidence for the importance of the C-terminal pathway for removal of the more basic ligands from Hg(II) before reaching the site of reduction.
The HgX2 studies also provided evidence for mobility in the C-terminal segment of the protein that may be important for catalysis. As alluded to above, the rate constants for Hg(II) binding to the CCAA enzyme were dramatically affected by the size of the ligand with delivery from Hg(CN)2 with small ligands being ca. 104-fold faster than from Hg(Cys)2 with bulky ligands. As seen in the space-filling model of the enzyme (Fig. 4), the C-terminal segment fills the cavity adjacent to the active site and, in this static picture, leaves only a narrow pathway to the inner cysteines too small to accommodate the bulky Hg(Cys)2 substrate if the C-terminal thiols are not present. Thus, for reaction of Hg(Cys)2 with the inner cysteines in the CCAA enzyme, this segment must move out of the way. Strict second-order kinetics observed in the reaction indicate that this motion is not rate-limiting but rather that the enzyme exists in an ensemble of conformational states most of which are closed to access by the bulky Hg(Cys)2 but are in rapid equilibrium with a small fraction that are open to access by this substrate. The 103-fold faster reaction of Hg(Cys)2 with the wild-type enzyme via the C-terminal cysteines over the direct reaction with the inner cysteines in CCAA  indicates that full extension of the segment out of the active site should not be necessary for catalysis. However, the depth of the accessible C-terminal cysteine in a surface cleft and the orientation of the two C-terminal cysteines in the structure  do suggest that mobility of the segment within the cavity may be important to bring the C-terminal cysteines closer to the surface for exchange with substrate (while Hg(0) escapes underneath) and then closer to the inner cysteines for delivery as depicted in Scheme 1.
Figure 4. Space-filling model of Bacillus sp. RC607 MerA. The main pathway is shown for entry of bulky Hg(SR)2 substrates via C-terminal cysteines and the alternative pathway that small non-physiological Hg(II) substrates such as HgBr2 and Hg(CN)2 can enter. The alternative pathway is too small for bulky Hg(SR)2 substrates, so the segment must move out of the way when the C-terminal cysteines are absent in the CCAA mutant.
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High-temperature factors in the crystal structure data at a hinge region of the segment are consistent with this prediction and also suggest that the motion toward the surface could bring the C-terminal cysteines closer to the amino group of a conserved lysine residue . With a similar pKa to thiol groups, this type of residue would be an appropriate choice to facilitate proton transfers from the enzyme thiols to the extraneous thiolate ligands during the initial steps of the exchange process. Additional residues would be expected to facilitate proton transfers between the enzyme thiols as Hg(II) is transferred down the path from the C-terminal to the inner cysteines. The only other conserved residues in this path are the two tyrosines identified as weak ligands to the Cd(II) in the crystal structure . In vivo analysis of TyrPhe mutations of these in the Bacillus enzyme indicates that although one tyrosine is more critical for activity, both are required for high-level resistance . Steady-state analysis again points to a lowered kcat/KM value as the most likely cause of the lost resistance, although both mutations also affect kcat[220,221].
Although the crystal structure of the Bacillus enzyme has proved invaluable for interpretation of the biochemical data, most of those data have been obtained through studies of the Tn501 enzyme, which previously failed to form diffractible crystals [206,222]. Although the sequences of the two proteins are 60–65% identical , the alignment indicates that some residues in the C-terminal segment and hypothetical binding pathway are not conserved. Thus before proceeding with further investigation of the properties of the C-terminal segment and residues in the binding pathway, the Miller lab has recently subcloned the catalytic core region of the Tn501 MerA (residues 96–561)  in an effort to obtain homogeneous protein free of the problematic proteolysis of the full-length protein . Both wild-type and mutant versions of this construct have been generated and found to diffract to 1.6 Å or better (Dong, Pai, Falkowski and Miller, unpublished results). Having the correct structure, particularly at high resolution, and a protein free of proteolytic problems will greatly benefit the design and interpretation of further structure–function analyses, including investigation of the apparent interactions between the active sites of the protein  that were difficult to explore using only structural information from the homologous protein. At present, detailed studies of the properties of the C-terminal segment and the role of residues in the binding pathway are a major focus of investigation in the Miller lab.
3.4.2NmerA – role of the N-terminal domain of MerA
As mentioned above, Brown et al.  hypothesized that since the N-terminal domain of MerA had no counterpart in the disulfide reductases, but included a pair of cysteines, it may serve a role in acquisition of Hg(II) from the transport protein(s). The discovery that approximately 70 amino acids of this domain were homologous in sequence to MerP, the periplasmic Hg(II) binding protein , enhanced this proposal. However, the observation that neither proteolysis of the first 85 N-terminal amino acids  nor site-directed mutagenesis of the N-terminal cysteines to alanines  had any effect on the in vitro catalytic properties of the core suggested that whatever role this domain had was not essential. The lack of effect of the Cys Ala mutations on the resistance phenotype appeared to corroborate this conclusion  and no further investigation of the properties or role of this domain was undertaken until very recently.
Several developments over the last decade have prompted a re-examination of the role of the N-terminal domain (NmerA). Well over 25 MerA sequences isolated from a wide variety of Gram-negative and Gram-positive bacteria can be found in a search of GenBank databases. Although a very small number of these lack an NmerA domain, the vast majority possess N-terminal regions with one or two repeats of the ∼70-amino acid domain (Fig. 5) indicating that this motif has been highly conserved through both vertical and horizontal transfer evolutionary processes. As mentioned in Section 3.2.1, both MerP and the NmerA domains are homologous with an increasing number of small soluble proteins and domains of soluble and membrane-bound proteins that have been identified as components of intracellular trafficking pathways for soft heavy metal ions such as Cu(I), Zn(II), Cd(II), and Hg(II). Recently solved structures of several of these cloned domains and proteins including MerP [24,226–231] show that they all adopt a similar ‘ferredoxin-like’βαββαβ structural fold  and utilize the cysteines of the conserved XXCXXC motif to chelate their cognate metal ions. Since NmerA should behave similarly, the structure of Hg-bound MerP can be used as a model to evaluate how it might interact with the catalytic core of MerA. As shown by the model in Fig. 6, the complementary shapes of the metal binding region of MerP and the cleft on the MerA core with the surface-accessible C-terminal cysteine provide a compelling argument for a role for the NmerA domain in delivery of Hg(II) to the core .
Figure 5. Clustal W, version 1.7 alignment of the N-terminal regions and initial portion of the catalytic core of 16 mercuric ion reductases. Sequences are from GenBank: Tn501, gi|66127|; Serratia marcescens, gi|126992|; Enterobacter cloacae, gi|2117123|; Alcaligenes sp., gi|2500120|; Tn21, gi|3513655|; Xanthomonas sp., gi|2120738|; Thiobacillus ferrooxidans, gi|126996|; Thiobacillus sp., gi|2765118|; Xanthomonas campestri, gi|6689529|; Shewanella putrefaciens, gi|2500123|; Bacillus sp. RC607, gi|126987|; Clostridium butyricum, gi|6177987|; Bacillus cereus, gi|2995409|; Exiguobacterium sp. gi|3413183|; Staphylococcus aureus, gi|126995|; Streptomyces lividans, gi|266529|. Conserved metal binding motif is bold, asterisks denote completely conserved residues.
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Figure 6. Model of core of Bacillus MerA  and Hg-bound MerP . Proteins are shown with MerP docked in trough where the surface-accessible C-terminal cysteine of MerA lies.
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To simplify analysis of the properties of the NmerA domain and its interactions with the catalytic core, the 69-amino acid N-terminal region and the catalytic core region including residues 96–561 of the Tn501 merA have recently been subcloned . The nearly completed NMR structure of NmerA shows it to be quite similar to its homologues with the expected βαββαβ fold (Ledwidge, Miller and Dötsch, unpublished results). As further expected, the protein binds a single equivalent of Hg(II) using the two cysteines of the XXCXXC motif, and the Hg(II) complex of the protein serves as an excellent substrate for the catalytic core in the absence of extraneous thiol-containing compounds, demonstrating that it definitely has the ability to serve as a facilitator for Hg(II) acquisition and delivery to the core (Ledwidge, Falkowski and Miller, unpublished results).
With these results in hand, functional analysis of the domain in the full-length protein has also been re-examined under conditions ensuring that both the N-terminal and C-terminal cysteines were fully reduced. Low thiol titers for the proteins assayed in the original comparisons [195,196] indicate that the N-terminal cysteines were not initially reduced when the proteins were added to the assays. This and the later observation that a disulfide at the N-terminus does not undergo reduction in the timeframe of the assays  suggest that the cysteines in the NmerA domain of the full-length protein may not have been available to participate in the reaction in those studies . In the new studies, steady-state profiles for fully reduced core and full-length proteins have been compared using cysteine and glutathione as small physiological thiol ligands for Hg(II), as well as E. coli thioredoxin as a protein dithiol chelator for Hg(II). The results show that the impact of the NmerA domain on the kinetics increases as the size of the thiol ligand on the Hg(II) substrate increases and as the excess concentration of the thiol ligand in the assay decreases (Ledwidge, Falkowski and Miller, unpublished results). This suggests that the NmerA domain may only become critical under conditions of depletion of the cellular thiol pool such that binding of Hg(II) to protein thiols would then become more problematic. Since the in vivo function of the N-terminal di-Ala mutant was tested  in otherwise unstressed E. coli, the normal intracellular thiol concentrations of GSH (ca. 6 mM) may have eliminated any benefit the NmerA domain could provide. This proposal is currently under investigation using GSH-depleted cells (Summers and Miller, unpublished results).
Another clue that the type and quantity of available thiol ligands may alter the impact of the domain on the reaction is provided by the species variation in the N-terminal appendage (Fig. 5). Comparison of the species from which MerAs have been isolated with published analyses of intracellular thiol content [233,234] suggests that the presence and number of repeats of the domain in the N-terminal appendage may be correlated with the major type and quantity of thiol synthesized in each species. Thus, cells that lack glutathione and have rather low intracellular concentrations of other thiols (some Bacillus and Clostridium sp.) have double repeats in their associated MerA appendages, while cells that synthesize glutathione (E. coli, Pseudomonas sp.) or have relatively high concentrations of other thiols such as cysteine or coenzyme A (S. aureus) have a single repeat in their MerA appendages. The correlation of double repeat sequences with low intracellular thiol concentration strongly suggests that the NmerA domains should be more critical for resistance in those organisms since they must serve both as the intracellular buffer to prevent inhibitory binding to other proteins in the cell and as the specific delivery agent to the catalytic core. This idea further suggests that in cells with higher concentrations of thiols, the single domain would not normally be needed as the buffering agent, but might become important in this role if the cells are subjected to additional oxidative or other stresses that deplete the intracellular thiol concentration. Note that the one sequence lacking an N-terminal domain in the list is from an organism that utilizes the unusual sugar derivative of cysteine, mycothiol, instead of glutathione as its cellular thiol . The implications of this curious observation require further investigation.
Kenzo Tonomura and his group first described the cell-free, substrate-inducible activity of a mercury-resistant soil Pseudomonas, strain K62, which degraded organomercurial compounds such as phenylmercury, ethylmercury, and methylmercury [79,235]. They posited a multi-protein complex involving one or more dehydrogenases and cytochromes as well as a specialized ‘decomposing enzyme’ and later characterized [191,192,236] two distinct organomercurial degrading activities from strain K62.
Simon Silver's group showed that inducible genes encoding organomercurial resistance occurred on transferrable plasmids in approximately 20% of mercury-resistant Enterobacteriaceae  and in all mercury-resistant Staphylococci. They also showed that in an E. coli carrying the conjugative IncM plasmid, R831, only two proteins were required for the organomercurial-degrading activity: a soluble enzyme which split the C–Hg bond in the organomercurial, releasing a protonated organic moiety (such as methane from methyl mercury, Fig. 2) and the Hg(II) cation which was then reduced to Hg(0) by the mercuric reductase, MerA  (for details on MerA, see above). The organomercurial-degrading enzyme whose size was estimated at 25 kDa was initially called a hydrolyase under the assumption that the proton added to the organic moiety arose from water. Inducible polypeptides in this size range were also observed in minicells programmed with plasmid R831 .
Meticulous characterization of the MerB encoded by R831 revealed it to have a molecular mass of 22.4 kDa, to lack any cofactors, and to function as a monomer [238,239]. The purified protein required a minimum two-fold molar excess of thiol over organomercurial substrate to exhibit any activity and preferred the physiological thiol cysteine to non-physiological mercaptans. The enzyme has a very broad substrate tolerance, handling both alkyl and aryl mercurials, with a slight preference for the latter. As is often the case with enzymes of low substrate specificity, MerB has very slow turnover rates ranging from 0.7 to 240 min−1 on various substrates. Although relatively slow for an enzyme, these rates do represent a 106–107-fold acceleration over chemical protonolysis rates of organomercurials. Paradoxically for a protonolysis catalyst, MerB's pH optimum was >9. Mechanistic studies revealed retention of the skeletal configuration of the substrate consistent with the rare SE2 mechanism rather than a radical-based mechanism. Deuterium isotope effects were consistent with a rate-limiting proton delivery step. Relevant chemical model studies  demonstrated a 1000-fold acceleration of aryl-Hg protonolysis by a compound capable of bis-coordination, whereas a monothiol reagent provided only a 50-fold rate acceleration, suggesting that in MerB two protein thiol groups might be involved in stabilizing a reaction intermediate.
Recent phylogenetic analysis makes it clear that MerB is a unique enzyme without known homologs which has evolved into several subgroups distinguishable on the basis of their primary and predicted secondary structures . Consistent with its carriage by transferrable plasmids, the MerB phylogeny does not map onto the phylogeny of the bacteria in which those sequenced examples have occurred, i.e. several examples of MerB found in distantly related Gram-positive and Gram-negative bacteria cluster closely and are equidistant from examples found in other Gram-positive and Gram-negative bacteria. Three cysteines are highly conserved at positions 96, 117, and 159 (numbering as for MerB of R831) and consistent with its high requirement for thiols, MerB is a cytosolic enzyme with no disulfide bonds. Cys96 and Cys159 are essential for catalysis and Cys117 appears to have a structural role rather than a catalytic role. MerB works equally well with either of the physiological thiols, glutathione or cysteine.
Most described Gram-positive mercury resistance operons are broad-spectrum (i.e. have one or more MerB genes; see Section 4). However, in Gram-negative bacteria the prevalence is closer to 20%. Broad-spectrum-resistance strains of both Gram-positive and Gram-negative bacteria frequently have two mer operons, a broad-spectrum locus and a narrow-spectrum locus, sometimes on the same plasmid as is the case with R831  and pDU1358 . Interestingly, in many occurrences the operon bearing the merB gene in broad-spectrum-resistant Enterobacteriaceae has undergone deletions of varying lengths removing most of the intervening genes between merB and the operonic promoter  resulting in the merB gene being much closer to that promoter. In these internally deleted operons, the associated merR gene is retained, possibly because the MerR protein of the co-resident narrow-spectrum resistance operon will not respond to organomercurials .
Fig. 7 is an updated reaction model embodying the current state of knowledge concerning MerB. In step 1, a cysteine (probably Cys159 ) of the fully reduced enzyme displaces the solvent thiol adduct from the organomercurial and a second protein cysteine (probably Cys96) forms a bis-coordinate structure with the aryl mercurial (step 2). The proton may be donated to this activated bis-coordinated complex by Cys96 or perhaps by some other protonated residue in MerB such as the highly conserved Tyr93. Once the protonated organic moiety leaves, MerB is stuck with Hg(II) (step 3) until two solvent monothiols can remove it (step 4). Interestingly, dithiothreitol (DTT) actually inhibits MerB, possibly by forming a stable three-coordinate complex with the product Hg(II) and one of MerB's cysteines. DTT inhibition can be slowly reversed by either cysteine or glutathione.
Figure 7. Roles of Thiols in MerB protonolysis of organomercurials. Small red numbers designate positions of cysteine residues in the primary structure of MerB of plasmid R831b . RSH is a low-molecular-mass, cytosolic thiol redox buffer such as glutathione. Ar refers to an aryl (aromatic) moiety in an organomercurial compound such as phenylmercuric acetate.
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While this model accounts for the minimum two-fold excess of thiol required for any catalytic turnover as observed by others, it does not explicitly address the paradoxically high (in vitro) pH optimum of MerB nor the requirement for at least a 15-fold molar excess of thiol for optimum activity [238,239,241].