Molecular Mimicry in Mercury Toxicology - American Chemical Society

Saskatoon, Saskatchewan S7N 0W9, Canada. ReceiVed December ... A widely accepted example is the transport of methylmercury-cysteine species, which are...
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Chem. Res. Toxicol. 2006, 19, 753-759

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Molecular Mimicry in Mercury Toxicology Ruth E. Hoffmeyer,† Satya P. Singh,† Christian J. Doonan,† Andrew R. S. Ross,‡ Richard J. Hughes,‡ Ingrid J. Pickering,† and Graham N. George*,† Department of Geological Sciences, UniVersity of Saskatchewan, 114 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada and National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada ReceiVed December 7, 2005

Molecular mimicry occurs when one molecular entity is “mistaken” for another by cellular or other biological processes, and is thought to arise from structural similarities between the two molecules in question. It has been postulated by others to be important in the mechanism of uptake of toxic metal species into living tissues. A widely accepted example is the transport of methylmercury-cysteine species, which are thought to mimic the amino acid methionine. We have used mass spectrometry and mercury LIII-edge X-ray absorption spectroscopy to understand the solution structure of complexes between methylmercury and cysteine. With a view to understanding the basis of the suggested molecular mimicry mechanisms, we have used computational chemistry to compare the structure of methionine with that of the dominant solution species L-cysteinato(methyl)mercury(II), and the structure of cystine with that of mercury(II) bis-L-cysteineate. We conclude that the structural similarities between metal compounds and natural products are insufficient to support a mechanism based on molecular mimicry, but instead, mechanisms involving a less-specific mimicry based on similarity with the LR region of the amino acid part of the molecule. Introduction Molecular mimicry occurs when one molecular entity is “mistaken” for another by cellular or other biological processes. This concept is important in the development of autoimmune responses with some infectious diseases. In these cases, antigenic determinants of the infectious microorganisms resemble structures in the tissue of the host, but differ enough to be recognized as foreign by the host immune system (1). A quite different molecular mimicry, of certain carbohydrates by specific peptides, has been suggested for possible drug use (2). Molecular mimicry is also thought to be an important mechanism by which toxic metal species are taken up into living tissues (3, 4), because of structural similarities between the two molecules in question. One well-accepted example is the transport of methylmercurycysteine species (5), which are thought to mimic the amino acid methionine, thereby gaining entry into the cell via an amino acid carrier, the LAT1 transporter (6). The structural similarity that has been supposed to exist between methionine and methylmercury-cysteine is shown in Figure 1. It has been assumed that molecular mimicry plays an important role in the toxicology of a number of different mercury species (4). Here, we critically examine the relationship between molecular form and structure and show that the structural similarity upon which molecular mimicry is thought to be based is, in many cases, an oversimplification.

Materials and Methods Sample Preparation. Reagents were purchased from Sigma Aldrich and STREM Chemical and were of the best available quality. Appropriate quantities of methylmercury hydroxide * To whom correspondence should be addressed. Phone, 306-966-5722; fax, 306-966-8593; e-mail, [email protected]. † University of Saskatchewan. ‡ National Research Council Canada, Plant Biotechnology Institute.

Figure 1. Schematic structures for (a) methionine, (b) L-cysteinato(methyl)mercury(II), (c) mercury(II) bis-L-cysteineate, and (d) cystine, showing structural similarities that have been posited between mercury compounds and natural products.

(CH3HgOH) and cysteine (Cys) were combined and incubated for 30 min at room temperature prior to spectroscopic examination. Samples for X-ray absorption spectroscopy (XAS) and electrospray ionization mass spectrometry (ESI-MS) were prepared using final Hg concentrations of 5 mM and 100 µM, respectively, in aqueous solution containing 50% (v/v) methanol. Methanol acted both as a glassing agent, to prevent artifacts from ice crystals during XAS, and an acceptable solvent for ESI-MS. Samples for XAS were loaded into 2 × 3 × 25 mm Lucite cuvettes and frozen in liquid nitrogen immediately prior to data collection. Crystalline L-cysteinato(methyl)mercury(II) (referred to below as methylmercury-cysteine) was prepared under dry N2 by mixing stoichiometric amounts of CH3HgOH and cysteine in 50% aqueous ethanol as described by Taylor et al. (5), evaporated under flowing dry N2, and then crystallized at 4 °C. Mass Spectrometry. Mass analysis of dissolved Hg species was carried out by negative-ion ESI-MS using a quadrupole tandem

10.1021/tx0503449 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/03/2006

754 Chem. Res. Toxicol., Vol. 19, No. 6, 2006 mass spectrometer (Quattro LC, Micromass, U.K.) fitted with a pneumatically assisted electrospray ion source (Z-spray, Micromass). Samples were introduced by flow injection using a binary solvent pump and autosampler (1100 series, Hewlett-Packard) fitted with a 10 µL sample loop and operating at a flow rate of 20 µL/ min. The carrier solvent consisted of 50:50 (v/v) methanol/water containing 1 mM ammonium acetate to maintain near-neutral pH, while ESI-MS parameters were adjusted to minimize dissociation of Hg species during analysis (7). X-ray Absorption Spectroscopy. Measurements were carried out at the Stanford Synchrotron Radiation Laboratory with the SPEAR storage ring containing 90-100 mA at 3.0 GeV. Mercury LIII-edge XAS data were collected on the structural molecular biology XAS beamline 9-3 with a wiggler field of 2 T, and employing a Si(220) double-crystal monochromator. Beamline 9-3 is equipped with a rhodium-coated vertical collimating mirror upstream of the monochromator, and a downstream bent-cylindrical focusing mirror (also rhodium-coated). Harmonic rejection was accomplished by setting the angle of the mirrors to correspond to a cutoff energy of 15 keV. Incident and transmitted X-ray intensities were monitored using nitrogen-filled ionization chambers. X-ray absorption was measured as the Hg LR1 fluorescence excitation spectrum using an array of 30 germanium detectors, with gallium filters, and a Soller slit assembly to preferentially reject scattered radiation (8-10). During data collection, samples were maintained at a temperature of approximately 10 K using a liquid helium flow cryostat. Between four and six 35-min scans were accumulated for each sample, and the absorption of a standard Hg-Sn amalgam metal foil was measured simultaneously by transmittance. The energy was calibrated with reference to the lowest energy LIII inflection point of the metal foil which was assumed to be 12285.0 eV. The spectrum of the Hg-Sn amalgam was determined to be identical with that of a micro-particulate aqueous suspension of pure metallic mercury (9). The extended X-ray absorption fine structure (EXAFS) oscillations χ(k) were quantitatively analyzed by curve-fitting using the EXAFSPAK suite of computer programs (11) as described by George et al. (12). Fourier transforms were phase-corrected for Hg-S backscattering. The threshold energy E0 was assumed to be 12305.0 eV, and ab initio theoretical phase and amplitude functions were calculated using the program FEFF version 8.2 (13, 14). Molecular Modeling. Density Functional Theory (DFT) molecular modeling used the program Dmol3 Materials Studio Version 3.2 (Accelrys, Inc.) (15, 16). DFT is a rigorous yet convenient and practical method for computing structural details of metal coordination compounds and has been extensively validated. We expect bond-length accuracies of better than 0.05 Å, and good estimates of energetic trends between postulated molecular entities. The Becke exchange (17) and Perdew correlation (18) functionals were used to calculate both the potential during the self-consistent field procedure and the energy. Double numerical basis sets included polarization functions for all atoms. Calculations were spinunrestricted, and all electron relativistic core potentials were used. No symmetry constraints were applied (unless otherwise stated), and optimized geometries used energy tolerances of 2.0 × 10-5 hartree. Detailed molecular comparisons were made using DS ViewerPro Suite 6.0 (Accelrys, Inc.) using the method of Hodgkin and Richards (19) which takes into account not only molecular shape, but also magnitudes of electron densities. To take into account both steric and electrostatic fields, the similarity index H was evaluated as an equally weighted sum of Hodgkin indexes (19) for the steric and electrostatic fields, with the latter from Mulliken atomic partial charges computed by Dmol3. Values for the similarity index in principle range from -1.0 to 1.0, with 1.0 indicating a perfect overlap between fields, zero indicating no overlap, and -1.0 only occurring with the electrostatic potential, with the molecules inversely aligned. This was tested using the small, very similar species, CH3OCH3, CH3SCH3, and CH3CH2CH3. For this series, values for the similarity index between 0 and 1 are reasonable due to their simplicity. The substitution of O for S and for CH2 introduces a change of approximately 1/3 of the volume, leaving

Hoffmeyer et al.

Figure 2. Different modes of mercury coordination. Computed structures are shown for coordination of mercuric ions by thiolates (ac) and of methylmercury by thiolates (d-f), showing digonal twocoordinate (a and d), trigonal three-coordinate (b and e), and approximately tetrahedral four-coordinate (c and f). For simplicity of computation and display, methyl groups are used to represent a generalized aliphatic group.

the remainder essentially unchanged. In comparing two molecules, the energy-minimized DFT coordinates were used as a starting point, and H was maximized by iteratively adjusting allowed conformations (e.g., torsion angles for rotatable bonds, etc.) for one or for both of the two molecules.

Results and Discussion Modes of Mercury Coordination. Mercury has a high affinity for thiols, and the existence of two-coordinate species containing one or more thiol ligands is often assumed in toxicological literature. Higher coordination numbers are almost never invoked, despite the fact that the chemistry of such species has been known for some years. Stable complexes are known with either two [Hg(SR)2], three [Hg(SR)3]-, or four [Hg(SR)4]2coordination, and well-characterized examples of all three coordination modes have been identified by X-ray crystallography (for example, see ref 20). A four-coordinate cysteine complex [Hg(Cysteine)4] has recently been reported to be formed in aqueous solutions at high pH values (21). Similar changes in mercury coordination by other ligands are wellknown. For example, aqueous mercuric chloride in the presence of excess Cl- can form the solutions species HgCl2, [HgCl3]-, and [HgCl4]2- with different levels of chloride. Figure 2 shows computed structures illustrating the three different categories of coordination of mercuric ions: digonal, trigonal planar, and tetrahedral. The metrical details of crystallographically characterized mercury-thiolate examples agree very well with our computed structures. Computed and crystallographic distances both show systematic increase in bond length with coordination number from 2.305 (two-coordinate) to 2.434 (three-coordinate) to 2.595 Å (four-coordinate). Fewer examples of methylmercury compounds have been characterized structurally, and to date, there is only crystallographic data available for digonally coordinated species (22),

Mercury Molecular Mimicry

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Figure 3. Mass spectrometry of dissolved methylmercury-cysteine compounds formed by mixing CH3HgOH (10 µM Hg, final concentration) with cysteine in 50:50 (v/v) methanol/water containing 1 mM (final) ammonium acetate to maintain near-neutral pH. Methylmercury/cysteine stoichiometries of solutions were (a) 1:1, (b) 1:5, and (c) 1:10. Methylmercury species are easily distinguished by the characteristic Hg isotope pattern.

including the methylmercury-cysteine compound HO2C(NH2)CHCH2S-Hg-CH3 (5) that features in the present study. Formal three-coordinate alkyl and aryl mercury species have been reported, and hitherto, all these compounds possess a T-shaped geometry comprising a digonally coordinated mercury and an elongated third bond. While bona fide four-coordinate species involving methylmercury have also been characterized (for example, see ref 23), none involves sulfur ligation to the metal. As expected from simple chemical considerations, our density functional calculations (Figure 2) predict that methylmercury species coordinated by one, two, or three thiolates will be stable entities and are therefore anticipated to form under favorable conditions. The computed bond lengths increase systematically with mercury coordination number, with HgsC bond lengths of 2.065, 2.152, and 2.197 Å, and HgsS bond lengths of 2.321, 2.463, and 2.665 Å, respectively. We have shown previously by comparison with test molecules of accurately known structure (9) that our density functional theory calculations consistently underestimate HgsS bond lengths by about 0.05 Å, and therefore, computed values of HgsS bond lengths can be corrected by simply offsetting by this amount, while computed HgsC bond lengths are more accurate. EXAFS has excellent accuracy in determining bond lengths (to better than 0.02 Å) and poorer accuracy for determining coordination numbers (ca. (25%). Thus, bond lengths can be used as an additional confirmation of the coordination number derived from curve-fitting. Methylmercury Coordination by Cysteine in Solution. Electrospray Ionization (ESI) mass spectrometry was used to study Hg coordination in solution, following addition of CH3HgOH to different concentrations of dissolved cysteine. Figure 3 shows the ESI mass spectrum obtained using cysteine/ CH3HgOH mole ratios of 1:1, 5:1, and 10:1, together with assignments for several of the peaks. Note that mercury species

are clearly identified by the characteristic distribution of Hg isotopes, and that mass-to-charge (m/z) values reported below refer solely to the most abundant (202Hg) isotope peaks. For example, the peak at m/z 336 corresponds to the anion [((CysH)‚HgCH3)-H]-,1 in which the cysteine is deprotonated (i.e., Cys-H) and therefore presumably bound covalently to Hg via sulfur. Deprotonation of the neutral, two-coordinate species [(Cys-H)‚HgCH3] in solution would result in the observed anion [((Cys-H)‚HgCH3)-H]-. The ESI mass spectrum also provides clear evidence for the formation of species of higher coordination number, with metal coordination similar to the structures shown in Figure 2. For example, the peak at m/z 576 corresponds to the singly charged anion [((Cys-H)3‚HgCH3)-H]-, in which all cysteines must be deprotonated (i.e., Cys-H) to produce an ion of the observed mass. In this case, however, loss of a single proton from the dissolved, four-coordinate species [(Cys-H)3‚ HgCH3]2- would result in an ion with a triple negative charge. This implies that two of the cysteines in the observed ion are joined by a disulfide bond, in much the same way that the cysteine-containing tripeptide glutathione can become oxidized to form a dimer while still binding a divalent metal ion (7). Hence, this ion may be more accurately represented as [((CysCys)(Cys-H)‚HgCH3)-H]-. Other species observed in the ESI mass spectrum apparently contain cysteines that are not deprotonated (i.e., Cys) and which therefore presumably interact noncovalently with methylmercury and/or cysteines to which the Hg is covalently bound. For example, the peak at m/z 491 corresponds to the ion [(Cys)2‚S‚ HgCH3]-, in which S is apparently derived from a covalently bound cysteine that underwent fragmentation during analysis. 1 In this section, we adopt the nomenclature often used in discussing mass spectrometry data of proteins and peptides in which Cys-H denotes a cysteine (e.g., HO2C(NH2)CHCH2SH) lacking a proton (e.g., HO2C(NH2)CHCH2S-), that is, minus H.

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Figure 4. Comparison of X-ray absorption spectra for dissolved methylmercury-cysteine species formed by mixing CH3HgOH (5 mM Hg, final concentration) with cysteine solutions to give stoichiometries of (a) 1:1, (b) 1:3, and (c) 1:10. (A) The Hg LIII near-edge spectra, and (B) the EXAFS Fourier transforms phase-corrected for HgsS backscattering. In both panels A and B, the spectra for the three different stoichiometries are seen to be essentially identical, indicating that the dominant solution species do not change with increasing levels of cysteine. The transform shows characteristic peaks at approximately 2.0 and 2.3 Å arising from HgsC and HgsS backscattering, respectively.

Detection of [S‚HgCH3]- (m/z 249) confirms fragmentation of cysteine and suggests a common precursor in [(Cys-H)‚ HgCH3]0, with noncovalent attachment of two cysteines giving rise to the ion at m/z 491. Partial decomposition of peptides and amino acids can occur in the ESI-MS interface, even under mild electrospray conditions (7). Cysteine is particularly susceptible to fragmentation, as confirmed by tandem MS experiments (not shown) in which selected species, including deprotonated cysteine ([Cys-H]-, m/z 120), were found to undergo collisionally activated decomposition at energies as low as 10 eV. Detection of [(Cys)‚HS‚CH3S‚ HgCH3]- (m/z 418), in which partial loss of two covalently bound cysteines is again balanced by the requisite number of protons, supports this hypothesis and implies the existence of a three-coordinate ionic species [(Cys-H)2‚HgCH3]- in solution, although surprisingly this particular ion (m/z 457) was not directly observed. Unfortunately partial decomposition of cysteine, combined with factors such as quadrupole transmission bias and different response factors for individual species, meant that we were unable to obtain accurate information on the relative amounts of these species using ESI-MS. Indeed, the solution species for which the MS data are most compelling is the two-coordinate complex with a single cysteine covalently bound to methylmercury. As a consequence, mercury LIII-edge X-ray absorption spectroscopy was employed in order to obtain quantitative information for Hg species. X-ray absorption spectra can be somewhat arbitrarily divided into two regions, the near-edge region, which is sensitive to electronic structure, and the extended X-ray absorption fine structure (EXAFS) oscillations, which can be used to determine details of local structure. Near-edge spectra provide a useful fingerprint of chemical type (10) but are difficult to interpret in quantitative molecular structural terms. In contrast, EXAFS spectra are relatively straightforward to interpret and provide accurate distances and identities of the nearby atoms. Figure 4 shows the Hg LIII X-ray absorption near-edge spectra and the EXAFS Fourier transforms of methylmercury in the presence of stoichiometric cysteine at pH 7.0, together with a 3-fold excess and a 10-fold excess of cysteine. The data clearly show that complexes with more than one cysteine bound to mercury do not form to an appreciable extent and that the majority solution species is the two-coordinate (Cys-H)HgCH3. This

Figure 5. EXAFS curve-fitting analysis of the data set shown in Figure 4c. (A) The experimental Hg LIII EXAFS oscillations (solid line) and best fit (broken line), and (B) the corresponding EXAFS Fourier transforms phase-corrected for HgsS backscattering. The best fit was calculated as described in the text and shown in Table 1.

indicates that the three-coordinate species identified by mass spectrometry must be a minor solution component. Quantitative analysis of the EXAFS spectra is illustrated in Figure 5. The analysis indicates a single HgsC bond at 2.07 Å and a single HgsS bond at 2.35 Å. Higher pH might favor increased coordination of mercury by cysteine (21), and because of this, we examined the XAS spectra with a 10-fold excess of cysteine as a function of pH, but two-coordinate species were detected up to pH 9.5 (not illustrated). The other parameters obtained from curve-fitting analysis are summarized in Table 1. Methylmercury-cysteine has been characterized by X-ray crystallography (5), and our EXAFS-derived bond lengths of the solution species are in excellent agreement both with the crystallographic values and with the results of analysis of the EXAFS spectrum of the crystalline solid (not illustrated).

Mercury Molecular Mimicry

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Table 1. EXAFS Curve-Fitting Results for Solution of L-Cysteinato(methyl)mercury(II)a interaction

N

R

σ2

∆E0

F

HgsC HgsS Hg‚‚‚Ob Hg‚‚‚Ob

1 1 2c 2c

2.070(3) 2.351(2) 3.27(1) 3.92(1)

0.0020(2) 0.0029(1) 0.0097(2) 0.0067(1)

-16.5(5)

0.2856

a Coordination numbers N, interatomic distances R are given in Å, Debye-Waller factors σ2 (the mean-square deviations in interatomic distance) in Å2, and the threshold energy shifts ∆E0 are given in eV. The values in parentheses are the estimated standard deviations obtained from the diagonal elements of the covariance matrix. The fit-error function F is

defined by F ) x∑k6(χ(k)calcd-χ(k)expt)2/∑χ(k)expt2, where χ(k) are the EXAFS oscillations and k is the photoelectron wavenumber given by k )

x2me/p2(E-E0). b Equivalent

fits were obtained with other atoms of similar atomic number (e.g., nitrogen or carbon). The most likely origin of these distant interactions is from weakly coordinated equatorial water ligands. Such interactions are commonly observed in solutions of mercuric species and in a number of crystallographically characterized species (22). c Only approximate coordination numbers are available for outer shell interactions.

Figure 6. Comparison of computed structures showing computed electron density isosurfaces mapped at 0.05 eÅ-3 of (a) methionine and (b) L-cysteinato (methyl)mercury(II).

Structural Similarity of L-Cysteinato(methyl)mercury(II) with Methionine and Other LAT1 Amino Acids. The computed energy-minimized molecular surfaces of methionine and methylmercury-cysteine are compared in Figure 6. The computed structure for methionine shows SsC bond lengths of 1.83 Å and a CsSsC bond angle of 100°. These compare well with the crystallographically determined values of 1.80 Å and 100° (24). As discussed above, it is well-established that two-coordinate mercury species are linear. A survey of the Cambridge Crystal Structure Database (22) for two-coordinate Hg2+ species with sulfur and carbon ligation indicates an average bond angle of 175(3)° and average HgsC and HgsS bond lengths of 2.07(4) and 2.37(2) Å, respectively. The computed structure of methylmercury-cysteine shows a CsHgsS bond angle of 177.4°, with HgsC and HgsS bond lengths of 2.06 and 2.34 Å, which compare well to the respective crystal-

lographic values for methylmercury-cysteine of 2.10 and 2.35 Å (5). It is clear from Figure 6 that the structural similarities between methylmercury-cysteine and methionine are patently superficial. The CsSsC bond angle of methionine is distinctly nonlinear at 100°, and its SsC bond lengths of 1.83 Å are significantly shorter than the HgsS bond of its purported molecular analogue. The chemical activity of a molecule is determined by more than just its shape; all chemical reactivity from chemical bond formation to hydrogen bonding is also dependent on the charge distribution within the molecule. Because of this, we have quantitatively evaluated molecular similarity by using a similarity index H that takes into account both molecular charge distribution and structural similarities. The molecules to be compared were overlaid to maximize the similarity indexes H between the structures. The energy-minimized structures from DFT (used to provide starting coordinates) were found to agree with available crystallographic data for the amino acids. In overlaying the structures, allowance was made for conformational flexibility expected in solution arising from bonds that can rotate, and the structural conformation was then refined to obtain the highest possible H. Possible values for H range from -1.0 to +1.0, but for the systems in the present study, it is not possible to get a condition corresponding to these conceptual limits of perfectly inversely aligned charge distribution or no overlap, so that values of H always tend to be close to +1.0. Thus, to characterize the expected range for H, we compared methionine to the seven other amino acids known to be transported by the LAT1 transporter. H varied from the least similar value of 0.79 for L-tryptophan to the most similar of 0.93 for L-histidine, with only tryptophan giving a value of H less than 0.86. The value for H for a case of genuine molecular mimicry should be very close to 1. For example, canavanine is a toxic amino acid present in Jack Beans that mimics arginine (25). This mimicry is the basis for canavanine’s biological activity and gives canavanine a similarity index H of 0.99 with arginine, irrespective of whether one molecule is allowed to vary, or both. The closest match of the structures of methylmercurycysteine and methionine when only the structure of methionine was allowed to adjust gave H ) 0.70, which represents a very poor match indeed. Figure 7 shows the structural superposition with the atoms colored according to partial charge. The figure shows the presence of both significant geometric and significant electrostatic mismatch in the region of the mercury, which is responsible for the poor similarity index. Indeed, H is lower in this case than the lowest value (for tryptophan) when comparing methionine with the other LAT1 amino acids. If both methionine and methylmercury-cysteine are allowed to vary, a value for H of 0.85 could be obtained. This is inferior to all but tryptophan in the methionine-LAT1 amino acid comparison. The similarity indices for methylmercury-cysteine and all the LAT1 amino acids are given in Table 2, where the coordinates for both molecules being compared were allowed to adjust. These comparisons clearly demonstrate that methylmercurycysteine shows little resemblance to methionine, nor does it resemble any of the other LAT1 amino acids. Methylmercurycysteine is thus not a molecular mimic of methionine or any of the other LAT1 amino acids. Structural Comparison of Cystine and Mercury(II) BisL-cysteineate. The computed molecular surfaces for the refined structures of cystine and mercury(II) bis-L-cysteineate are compared in Figure 8. Crystal structures are available for cystine (26) and for related Hg2+ conjugates of cysteine, such as the

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Figure 7. Calculated molecular superposition of methionine and L-cysteinato (methyl)mercury(II) showing the closest molecular match of methionine to L-cysteinato (methyl)mercury(II). The atoms are colored according to Mulliken partial atomic charge computed from Density Functional Theory. The arrows indicate the rotational degrees of freedom used in matching the two structures. Clear mismatch between the two species is evident from both structural and electrostatic perspectives.

Figure 9. Calculated molecular superposition of cystine and mercury(II) bis-L-cysteineate showing the closest molecular match. The atoms are colored according to Mulliken partial atomic charge computed from Density Functional Theory. The arrows indicate the rotational degrees of freedom used in matching the two structures. Clear mismatch between the two species is evident from both structural and electrostatic perspectives.

and the Hg2+ cysteine conjugate show little structural similarity, and the similarity index between molecules fitted to each other is only 0.86. This closest match is shown in Figure 9. Examination of the figure indicates that the two LR amino acid regions of mercury di-cysteinate are well and partly aligned, respectively. The charge variation of the SsHgsS region is largely responsible for keeping the similarity index constant, even though with two LR regions it might be expected to increase. In any case, it is clear that the Hg2+ cysteine conjugate cannot be a simple molecular mimic of cystine. The view of molecular structure used in the present work contrasts with the picture that is often shown to justify molecular mimicry. In these cases, the chemistry is essentially ignored, and the mercury is often depicted as having close to tetrahedral bond angles (i.e., ∼109°) and short bond lengths to its ligands (4, 28-31).

Conclusions

Figure 8. Comparison of computed structures showing computed electron density isosurfaces mapped at 0.05 eÅ-3 for (a) cystine and (b) mercury(II) bis-L-cysteineate. Table 2. Molecular Similarities for LAT1 Amino Acids and L-Cysteinato(methyl)mercury(II) compound L-isoleucine L-leucine L-valine L-histidine L-phenylalanine L-tryptophan L-tyrosine L-methionine

L-cysteinato(methyl)mercury(II)

L-methionine

0.840494 0.820970 0.864424 0.824209 0.811349 0.808089 0.867366 0.851896

0.919358 0.902727 0.880155 0.929818 0.908022 0.787094 0.863290 1.0

T-shaped species [(Cys)S]2Hg-Cl (27), and these show structural similarities to our computed structures. Our calculations indicate a number of alternative stable structures for the mercury di-cysteinate molecule, for example, a T-shaped species with an oxygen atom at ∼3.5 Å from mercury. Similar to methionine and methylmercury-cysteine, the molecular surfaces of cystine

Our analysis indicates that the molecular bases of many of the proposed molecular mimicry mechanisms in mercury toxicology are untenable and not based on detailed chemical knowledge. It has been conclusively shown that, in the presence of excess cysteine, methylmercury is taken up by the LAT1 transporter (6). However, it seems clear from the structural differences of the two compounds that this is not based upon a specific structural similarity between methionine and methylmercury, and a broader based mimicry in which the transporter recognizes the amino acid component of the molecule may be more relevant to the mechanism. Indeed, higher values of similarity index between methylmercury-cysteine and the LAT1 amino acids occurred only when the molecular overlay resulted in alignment of the LR region. Thus, while molecular mimicry cannot be responsible for the activity of LAT1 toward methylmercury-cysteine, this might be explained as mimicry of a molecular fragment. The best-established and perhaps most valid examples of molecular mimicry in inorganic chemical toxicology are those oxy-anions suggested as molecular mimics in the pioneering work of Wetterhahn-Jennette (3). However, even with these, a recent review of toxic metal transport by molecular mimicry showed flawed chemistry (4). This work not only depicted a number of mercury coordination compounds with incorrect coordination geometry (as we have discussed here), but also depicted incorrect molecular geometries for simple anions such as sulfate, selenate, and molybdate which

Mercury Molecular Mimicry

are shown as square-based pyramids, rather than as tetrahedral species. The models that are constructed to accurately describe the biochemistry of toxic metals must be as rigorous, at all levels, as the limits of our applied techniques. Furthermore, it is imperative that a chemically accurate description of metals and their ligands be retained as a central premise of any such model. Acknowledgment. This work was supported by a grant from the Canadian Institutes of Health Research. Research at the University of Saskatchewan was supported by Canada Research Chair awards (to G.N.G. and I.J.P.), the University of Saskatchewan, the Province of Saskatchewan, the National Institutes of Health, and the Natural Sciences and Engineering Research Council (Canada). Funding for the acquisition and operation of mass spectrometry equipment at the Plant Biotechnology Institute was provided by the Agri-Food Innovation Fund and the National Research Council of Canada, respectively. Portions of this work were also carried out at the Stanford Synchrotron Radiation Laboratory which is funded by the U.S. Department of Energy, Office of Basic Energy Sciences and Office of Biological and Environmental Sciences, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program.

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(10) (11) (12)

(13) (14) (15) (16) (17) (18) (19) (20)

Supporting Information Available: Tables of density functional theory computed-coordinates for methionine, L-cysteinato(methyl)mercury(II), cystine, and mercury(II) bis-L-cysteineate. This material is available free of charge via the Internet at http:// pubs.acs.org.

(21) (22)

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