Comment on "New insights into the biomineralization of mercury selenide nanoparticles through stable isotope analysis in giant petrel tissues".

Some birds and cetaceans can demethylate the toxic methylmercury cysteinate (MeHgCys) complex into inert mercury sulfide (HgSe) through the formation of an intermediate tetrahedral selenolate complex with selenocysteine (Sec) residues (Hg(Sec)4). The nucleation of the HgSe biominerals involves the substitution of the Se ligand for the Sec residues, which is considered to occur in the form of multinuclear Hgx(Se,Sec)y clusters mediated by proteins. Queipo-Abad et al. (2022) isolated HgSe nanoparticles from the biological tissues of giant petrels and measured the mass-dependent fractionation of the 202Hg isotope (δ202Hg). They concluded that the δ202Hg values of the HgSe nanoparticles from each tissue of individual petrels are specific to the HgSe species alone and that the Hg(Sec)4 → HgSe reaction occurs without fractionation of the 202Hg isotope. We show (1) that the HgSe nanoparticles are likely mixtures of MeHgCys, Hg(Sec)4, and HgSe, and therefore that the δ202Hg values are not species-specific, and (2) that the 202Hg isotope is actually fractionated during the Hg(Sec)4 → HgSe reaction, and therefore that this isotope can be used to trace the Hg metabolic pathways between tissues in a single individual and in different animals.

Chemical Information in the L3 X-ray Absorption Spectra of Molybdenum Compounds by High-Energy-Resolution Detection and Density Functional Theory.

X-ray spectroscopy using high-energy-resolution fluorescence detection (HERFD) has critically increased the information content in X-ray spectra. We extend this technique to the tender X-ray range and present a study at the L3-edge of molybdenum. We show how information on the oxidation state, phase composition, and local environment in molybdenum-based compounds can be obtained by analyzing the HERFD L3 X-ray absorption near-edge structure (XANES). We demonstrate that the chemical shift of the L3-edge HERFD spectra follows a parabolic dependence on the oxidation state and show that a qualitative analysis of high-resolution spectra can help to estimate parameters such as distortion of a ligand environment and radial order of atoms around the absorber. In certain cases, the spectra allow disentangling the contributions from bond lengths and angles to the distortion of the ligand polyhedron. Comparison of the high-resolution spectra with theoretical simulations shows that the single-electron approximation is able to reproduce the spectral shape. The results of this work may be useful in every branch of physics, inorganic and organometallic chemistry, catalysis, materials science, biochemistry, and mineralogy where observed changes in performance or chemical properties of Mo-based compounds, accompanied by small changes in spectral shape, are to be related to the details of electronic structure and local atomic environment.

Mercury Isotope Fractionation by Internal Demethylation and Biomineralization Reactions in Seabirds: Implications for Environmental Mercury Science.

A prerequisite for environmental and toxicological applications of mercury (Hg) stable isotopes in wildlife and humans is quantifying the isotopic fractionation of biological reactions. Here, we measured stable Hg isotope values of relevant tissues of giant petrels (Macronectes spp.). Isotopic data were interpreted with published HR-XANES spectroscopic data that document a stepwise transformation of methylmercury (MeHg) to Hg-tetraselenolate (Hg(Sec)4) and mercury selenide (HgSe) (Sec = selenocysteine). By mathematical inversion of isotopic and spectroscopic data, identical δ202Hg values for MeHg (2.69 ± 0.04‰), Hg(Sec)4 (-1.37 ± 0.06‰), and HgSe (0.18 ± 0.02‰) were determined in 23 tissues of eight birds from the Kerguelen Islands and Adélie Land (Antarctica). Isotopic differences in δ202Hg between MeHg and Hg(Sec)4 (-4.1 ± 0.1‰) reflect mass-dependent fractionation from a kinetic isotope effect due to the MeHg → Hg(Sec)4 demethylation reaction. Surprisingly, Hg(Sec)4 and HgSe differed isotopically in δ202Hg (+1.6 ± 0.1‰) and mass-independent anomalies (i.e., changes in Δ199Hg of ≤0.3‰), consistent with equilibrium isotope effects of mass-dependent and nuclear volume fractionation from Hg(Sec)4 → HgSe biomineralization. The invariance of species-specific δ202Hg values across tissues and individual birds reflects the kinetic lability of Hg-ligand bonds and tissue-specific redistribution of MeHg and inorganic Hg, likely as Hg(Sec)4. These observations provide fundamental information necessary to improve the interpretation of stable Hg isotope data and provoke a revisitation of processes governing isotopic fractionation in biota and toxicological risk assessment in wildlife.

Cellular remains in a ~3.42-billion-year-old subseafloor hydrothermal environment.

Subsurface habitats on Earth host an extensive extant biosphere and likely provided one of Earth's earliest microbial habitats. Although the site of life's emergence continues to be debated, evidence of early life provides insights into its early evolution and metabolic affinity. Here, we present the discovery of exceptionally well-preserved, ~3.42-billion-year-old putative filamentous microfossils that inhabited a paleo-subseafloor hydrothermal vein system of the Barberton greenstone belt in South Africa. The filaments colonized the walls of conduits created by low-temperature hydrothermal fluid. Combined with their morphological and chemical characteristics as investigated over a range of scales, they can be considered the oldest methanogens and/or methanotrophs that thrived in an ultramafic volcanic substrate.

Acute Toxicity of Divalent Mercury to Bacteria Explained by the Formation of Dicysteinate and Tetracysteinate Complexes Bound to Proteins in Escherichia coli and Bacillus subtilis.

Bacteria are the most abundant organisms on Earth and also the major life form affected by mercury (Hg) poisoning in aquatic and terrestrial food webs. In this study, we applied high energy-resolution X-ray absorption near edge structure (HR-XANES) spectroscopy to bacteria with intracellular concentrations of Hg as low as 0.7 ng/mg (ppm) for identifying the intracellular molecular forms and trafficking pathways of Hg in bacteria at environmentally relevant concentrations. Gram-positive Bacillus subtilis and Gram-negative Escherichia coli were exposed to three Hg species: HgCl2, Hg-dicysteinate (Hg(Cys)2), and Hg-dithioglycolate (Hg(TGA)2). In all cases, Hg was transformed into new two- and four-coordinate cysteinate complexes, interpreted to be bound, respectively, to the consensus metal-binding CXXC motif and zinc finger domains of proteins, with glutathione acting as a transfer ligand. Replacement of zinc cofactors essential to gene regulatory proteins with Hg would inhibit vital functions such as DNA transcription and repair and is suggested to be a main cause of Hg genotoxicity.

Demethylation of Methylmercury in Bird, Fish, and Earthworm.

Toxicity of methylmercury (MeHg) to wildlife and humans results from its binding to cysteine residues of proteins, forming MeHg-cysteinate (MeHgCys) complexes that hinder biological functions. MeHgCys complexes can be detoxified in vivo, yet how this occurs is unknown. We report that MeHgCys complexes are transformed into selenocysteinate [Hg(Sec)4] complexes in multiple animals from two phyla (a waterbird, freshwater fish, and earthworms) sampled in different geographical areas and contaminated by different Hg sources. In addition, high energy-resolution X-ray absorption spectroscopy (HR-XANES) and chromatography-inductively coupled plasma mass spectrometry of the waterbird liver support the binding of Hg(Sec)4 to selenoprotein P and biomineralization of Hg(Sec)4 to chemically inert nanoparticulate mercury selenide (HgSe). The results provide a foundation for understanding mercury detoxification in higher organisms and suggest that the identified MeHgCys to Hg(Sec)4 demethylation pathway is common in nature.

In Vivo Formation of HgSe Nanoparticles and Hg-Tetraselenolate Complex from Methylmercury in Seabirds-Implications for the Hg-Se Antagonism.

In vivo and in vitro evidence for detoxification of methylmercury (MeHg) as insoluble mercury selenide (HgSe) underlies the central paradigm that mercury exposure is not or little hazardous when tissue Se is in molar excess (Se:Hg > 1). However, this hypothesis overlooks the binding of Hg to selenoproteins, which lowers the amount of bioavailable Se that acts as a detoxification reservoir for MeHg, thereby underestimating the toxicity of mercury. This question was addressed by determining the chemical forms of Hg in various tissues of giant petrels Macronectes spp. using a combination of high energy-resolution X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopy, and transmission electron microscopy coupled to elemental mapping. Three main Hg species were identified, a MeHg-cysteinate complex, a four-coordinate selenocysteinate complex (Hg(Sec)4), and a HgSe precipitate, together with a minor dicysteinate complex Hg(Cys)2. The amount of HgSe decreases in the order liver > kidneys > brain = muscle, and the amount of Hg(Sec)4 in the order muscle > kidneys > brain > liver. On the basis of biochemical considerations and structural modeling, we hypothesize that Hg(Sec)4 is bound to the carboxy-terminus domain of selenoprotein P (SelP) which contains 12 Sec residues. Structural flexibility allows SelP to form multinuclear Hgx(Se,Sec)y complexes, which can be biomineralized to HgSe by protein self-assembly. Because Hg(Sec)4 has a Se:Hg molar ratio of 4:1, this species severely depletes the stock of bioavailable Se for selenoprotein synthesis and activity to one µg Se/g dry wet in the muscle of several birds. This concentration is still relatively high because selenium is naturally abundant in seawater, therefore it probably does not fall below the metabolic need for essential selenium. However, this study shows that this may not be the case for terrestrial animals, and that muscle may be the first tissue potentially injured by Hg toxicity.

Mercury in the tissues of five cephalopods species: First data on the nervous system.

Mercury (Hg), one of the elements most toxic to biota, accumulates within organisms throughout their lifespan and biomagnifies along trophic chain. Due to their key role in marine systems, cephalopods constitute a major vector of Hg in predators. Further, they grow rapidly and display complex behaviours, which can be altered by neurotoxic Hg. This study investigated Hg concentrations within 81 cephalopod specimens sampled in the Bay of Biscay, which belonged to five species: Eledone cirrhosa, Sepia officinalis, Loligo vulgaris, Todaropsis eblanae and Illex coindetii. Hg concentrations were measured in the digestive gland, the mantle muscle and the optic lobes of the brain. The digestive gland and the mantle were tissues with the most concentrated Hg among all species considered (up to 1.50 µg.g-1 dw), except E. cirrhosa. This benthic cephalopod had 1.3-fold higher Hg concentrations in the brain (up to 1.89 µg.g-1 dw) than in the mantle, while other species had 2-fold lower concentrations of Hg in the brain than in the mantle. Brain-Hg concentrations can be predicted from muscle-Hg concentrations for a given species, which facilitates the assessment of Hg toxicokinetics in cephalopods. In the most contaminated E. cirrhosa individual, the chemical form of Hg in its digestive gland, mantle muscle and optic lobes, was determined using High energy-Resolution X-ray Absorption Near Edge Structure (HR XANES) spectroscopy. In the digestive gland, 33 ± 11% of total Hg was inorganic Hg speciated as a dicysteinate complex (Hg(Cys)2), which suggested that the demethylation of dietary MeHg occurs in this organ. All Hg found in the mantle muscle and the optic lobes is methylated and bound to one cysteinyl group (MeHgCys complex), which implies that dietary MeHg is distributed to these tissues via the bloodstream. These results raised the questions regarding interspecific differences observed regarding Hg brain concentrations and the possible effect of Hg on cephalopod functional brain plasticity and behaviour.

TEXS: in-vacuum tender X-ray emission spectrometer with 11 Johansson crystal analyzers.

The design and first results of a large-solid-angle X-ray emission spectrometer that is optimized for energies between 1.5 keV and 5.5 keV are presented. The spectrometer is based on an array of 11 cylindrically bent Johansson crystal analyzers arranged in a non-dispersive Rowland circle geometry. The smallest achievable energy bandwidth is smaller than the core hole lifetime broadening of the absorption edges in this energy range. Energy scanning is achieved using an innovative design, maintaining the Rowland circle conditions for all crystals with only four motor motions. The entire spectrometer is encased in a high-vacuum chamber that allocates a liquid helium cryostat and provides sufficient space for in situ cells and operando catalysis reactors.

The chemical species of mercury accumulated by Pseudomonas idrijaensis, a bacterium from a rock of the Idrija mercury mine, Slovenia.

A mercury-resistant bacterial strain has been isolated from a rock of the Idrija mercury mine in Slovenia. The rock had 19 g carbon and 2952 mg mercury (Hg) per kg. Mass spectrometry and DNA sequencing showed that the bacterium belongs to the Pseudomonas genus. It is called Pseudomonas idrijaensis. This bacterial strain is sensitive to methylmercury (MeHg) like the reference P. aeruginosa strain PAO1, and is resistant to divalent mercury (Hg(II)) in contrast to PAO1. This difference could be attributed to the presence of the mer operon yet deprived of the merB gene encoding the organomercurial lyase, on the basis of whole genome sequencing. The P. idrijaensis mer operon displays the RTPCADE organization and is contained in the Tn5041 transposon. This transposon identified here occurs in other Gram-negative Hg-resistant strains isolated from mercury ores, aquatic systems and soils, including Pseudomonas strains from 15,000 to 40,000 years old Siberian permafrost. When P. idrijaensis was exposed to mercury chloride, two intracellular Hg species were identified by high energy-resolution XANES spectroscopy, a dithiolate Hg(SR)2 and a tetrathiolate Hg(SR)4 complex. P. idrijaensis had a much higher [Hg(SR)2]/[Hg(SR)4] molar ratio than bacteria lacking the mer operon when exposed to 4 µg Hg2+/L - resulting in an intracellular accumulation of 4.3 µg Hg/g dw. A higher amount of the Hg(SR)2 complex provides a chemical signature for the expression of the dicysteinate Mer proteins in response to mercury toxicity.

Divalent Mercury in Dissolved Organic Matter Is Bioavailable to Fish and Accumulates as Dithiolate and Tetrathiolate Complexes.

The freshwater cyprinid Tanichthys albonubes was used to assess the bioavailability of divalent mercury (Hg(II)) complexed in dissolved organic matter (DOM) to fish. The fish acquired 0.3 to 2.2 µg Hg/g dry weight after 8 weeks in aquaria containing DOM from a Carex peat with complexed mercury at initial concentrations of 14 nM to 724 nM. Changes in the relative proportions of dithiolate Hg(SR)2 and nanoparticulate ß-HgS in the DOM, as quantified by high energy-resolution XANES (HR-XANES) spectroscopy, indicate that Hg(SR)2 complexes either produced by microbially induced dissolution of nanoparticulate ß-HgS in the DOM or present in the original DOM were the forms of mercury that entered the fish. In the fish with 2.2 µg Hg/g, 84 ± 8% of Hg(II) was bonded to two axial thiolate ligands and one or two equatorial N/O electron donors (Hg[(SR)2+(N/O)1-2] coordination), and 16% had a Hg(SR)4 coordination, as determined by HR-XANES. For comparison, fish exposed to Hg2+ from 40 nM HgCl2 contained 10.4 µg Hg/g in the forms of dithiolate (20 ± 10%) and tetrathiolate (23 ± 10%) complexes, and also Hg xS y clusters (57 ± 15%) having a ß-HgS-type local structure and a dimension that exceeded the size of metallothionein clusters. There was no evidence of methylmercury in the fish or DOM within the 10% uncertainty of the HR-XANES. Together, the results indicate that inorganic Hg(II) bound to DOM is a source of mercury to biota with dithiolate Hg(SR)2 complexes as the immediate species bioavailable to fish, and that these complexes transform in response to cellular processes.

Mercury(II) Binding to Metallothionein in Mytilus edulis revealed by High Energy-Resolution XANES Spectroscopy.

Of all divalent metals, mercury (HgII ) has the highest affinity for metallothioneins. HgII is considered to be enclosed in the α and ß domains as tetrahedral α-type Hg4 Cys11-12 and ß-type Hg3 Cys9 clusters similar to CdII and ZnII . However, neither the four-fold coordination of Hg nor the existence of Hg-Hg atomic pairs have ever been demonstrated, and the HgII partitioning among the two protein domains is unknown. Using high energy-resolution XANES spectroscopy, MP2 geometry optimization, and biochemical analysis, evidence for the coexistence of two-coordinate Hg-thiolate complex and four-coordinate Hg-thiolate cluster with a metacinnabar-type (ß-HgS) structure in the α domain of separate metallothionein molecules from blue mussel under in vivo exposure is provided. The findings suggest that the CXXC claw setting of thiolate donors, which only exists in the α domain, acts as a nucleation center for the polynuclear complex and that the five CXC motifs from this domain serve as the cluster-forming motifs. Oligomerization is driven by metallophilic Hg⋅⋅⋅Hg interactions. Our results provide clues as to why Hg has higher affinity for the α than the ß domain. More generally, this work provides a foundation for understanding how metallothioneins mediate mercury detoxification in the cell under in vivo conditions.

Chemical Forms of Mercury in Pyrite: Implications for Predicting Mercury Releases in Acid Mine Drainage Settings.

Pyrite (cubic FeS2) is the most abundant metal sulfide in nature and also the main host mineral of toxic mercury (Hg). Release of mercury in acid mine drainage resulting from the oxidative dissolution of pyrite in coal and ore and rock resulting from mining, processing, waste management, reclamation, and large construction activities is an ongoing environmental challenge. The fate of mercury depends on its chemical forms at the point source, which in turn depends on how it occurs in pyrite. Here, we show that pyrite in coal, sedimentary rocks, and hydrothermal ore deposits can host varying structural forms of Hg which can be identified with high energy-resolution XANES (HR-XANES) spectroscopy. Nominally divalent Hg is incorporated at the Fe site in pyrite from coal and at a marcasite-type Fe site in pyrite from sedimentary rocks. Distinction of the two Hg bonding environments offers a mean to detect microscopic marcasite inclusions (orthorhombic FeS2) in bulk pyrite. In epigenetic pyrite from Carlin-type Au deposit, up to 55 ± 6 at. % of the total Hg occurs as metacinnabar nanoparticles (ß-HgSNP), with the remainder being substitutional at the Fe site. Pyritic mercury from Idrija-type Hg deposit (α-HgS ore) is partly divalent and substitutional and partly reduced into elemental form (liquid). Divalent mercury ions, mercury sulfide nanoparticles, and elemental mercury released by the oxidation of pyrite in acid mine drainage settings would have different environmental pathways. Our results could find important applications for designing control strategies of mercury released to land and water in mine-impacted watersheds.

Biogenesis of Mercury-Sulfur Nanoparticles in Plant Leaves from Atmospheric Gaseous Mercury.

Plant leaves serve both as a sink for gaseous elemental mercury (Hg) from the atmosphere and a source of toxic mercury to terrestrial ecosystems. Litterfall is the primary deposition pathway of atmospheric Hg to vegetated soils, yet the chemical form of this major Hg input remains elusive. We report the first observation of in vivo formation of mercury sulfur nanoparticles in intact leaves of 22 native plants from six different species across two sampling areas from China. The plants grew naturally in soils from a mercury sulfide mining and retorting region at ambient-air gaseous-Hg concentrations ranging from 131 ± 19 to 636 ± 186 ng m-3 and had foliar Hg concentration between 1.9 and 31.1 ng Hg mg-1 dry weight (ppm). High energy resolution X-ray absorption near-edge structure (HR-XANES) spectroscopy shows that up to 57% of the acquired Hg is nanoparticulate, and the remainder speciated as a bis-thiolate complex (Hg(SR)2). The fractional amount of nanoparticulate Hg is not correlated with Hg concentration. Variation likely depends on leaf age, plant physiology, and natural variability. Nanoparticulate Hg atoms are bonded to four sulfide or thiolate sulfur atoms arranged in a metacinnabar-type (ß-HgS) coordination environment. The nanometer dimension of the mercury-sulfur clusters outmatches the known binding capacity of plant metalloproteins. These findings give rise to challenging questions on their exact nature, how they form, and their biogeochemical reactivity and fate in litterfall, whether this mercury pool is recycled or stored in soils. This study provides new evidence that metacinnabar-type nanoparticles are widespread in oxygenated environments.

Mercury Trithiolate Binding (HgS3) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains.

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS3 coordination). The model cyclodecapeptide P3C, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS3 coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P2C cyclodecapeptide validated previously as a model for d10 metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP3C complex with a HgS3 coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and 199Hg NMR. Hg LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP3C complex was refined based on 1H-1H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgP3CH complex has a p Ka of 4.3, indicating that the HgS3 coordination mode is stable over a large range of pH. This low p Ka value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P3C.

High energy-resolution x-ray spectroscopy at ultra-high dilution with spherically bent crystal analyzers of 0.5 m radius.

We present the development, manufacturing, and performance of spherically bent crystal analyzers (SBCAs) of 100 mm diameter and 0.5 m bending radius. The elastic strain in the crystal wafer is partially released by a "strip-bent" method where the crystal wafer is cut into strips prior to the bending and the anodic bonding process. Compared to standard 1 m SBCAs, a gain in intensity is obtained without loss of energy resolution. The gain ranges between 2.5 and 4.5, depending on the experimental conditions and the width of the emission line measured. This reduces the acquisition times required to perform high energy-resolution x-ray absorption and emission spectroscopy on ultra-dilute species, accessing concentrations of the element of interest down to, or below, the ppm (ng/mg) level.

Nucleation of mercury sulfide by dealkylation.

Metal sulfide minerals are assumed to form naturally at ambient conditions via reaction of a metallic element with (poly)sulfide ions, usually produced by microbes in oxygen-depleted environments. Recently, the formation of mercury sulfide (ß-HgS) directly from linear Hg(II)-thiolate complexes (Hg(SR)2) in natural organic matter and in cysteine solutions was demonstrated under aerated conditions. Here, a detailed description of this non-sulfidic reaction is provided by computations at a high level of molecular-orbital theory. The HgS stoichiometry is obtained through the cleavage of the S-C bond in one thiolate, transfer of the resulting alkyl group (R') to another thiolate, and subsequent elimination of a sulfur atom from the second thiolate as a thioether (RSR'). Repetition of this mechanism leads to the formation of RS-(HgS)n-R chains which may self-assemble in parallel arrays to form cinnabar (α-HgS), or more commonly, quickly condense to four-coordinate metacinnabar (ß-HgS). The mechanistic pathway is thermodynamically favorable and its predicted kinetics agrees with experiment. The results provide robust theoretical support for the abiotic natural formation of nanoparticulate HgS under oxic conditions and in the absence of a catalyst, and suggest a new route for the (bio)synthesis of HgS nanoparticles with improved technological properties.

Chemical Forms of Mercury in Human Hair Reveal Sources of Exposure.

Humans are contaminated by mercury in different forms from different sources. In practice, contamination by methylmercury from fish consumption is assessed by measuring hair mercury concentration, whereas exposure to elemental and inorganic mercury from other sources is tested by analysis of blood or urine. Here, we show that diverse sources of hair mercury at concentrations as low as 0.5 ppm can be individually identified by specific coordination to C, N, and S ligands with high energy-resolution X-ray absorption spectroscopy. Methylmercury from seafood, ethylmercury used as a bactericide, inorganic mercury from dental amalgams, and exogenously derived atmospheric mercury bind in distinctive intermolecular configurations to hair proteins, as supported by molecular modeling. A mercury spike located by X-ray nanofluorescence on one hair strand could even be dated to removal of a single dental amalgam. Chemical forms of other known or putative toxic metals in human tissues could be identified by this approach with potential broader applications to forensic, energy, and materials science.

Structure, Bonding, and Stability of Mercury Complexes with Thiolate and Thioether Ligands from High-Resolution XANES Spectroscopy and First-Principles Calculations.

We present results obtained from high energy-resolution L3-edge XANES spectroscopy and first-principles calculations for the structure, bonding, and stability of mercury(II) complexes with thiolate and thioether ligands in crystalline compounds, aqueous solution, and macromolecular natural organic matter (NOM). Core-to-valence XANES features that vary in intensity differentiate with unprecedented sensitivity the number and identity of Hg ligands and the geometry of the ligand environment. Post-Hartree-Fock XANES calculations, coupled with natural population analysis, performed on MP2-optimized Hg[(SR)2···(RSR)n] complexes show that the shape, position, and number of electronic transitions observed at high energy-resolution are directly correlated to the Hg and S (l,m)-projected empty densities of states and occupations of the hybridized Hg 6s and 5d valence orbitals. Linear two-coordination, the most common coordination geometry in mercury chemistry, yields a sharp 2p to 6s + 5d electronic transition. This transition varies in intensity for Hg bonded to thiol groups in macromolecular NOM. The intensity variation is explained by contributions from next-nearest, low-charge, thioether-type RSR ligands at 3.0-3.3 Å from Hg. Thus, Hg in NOM has two strong bonds to thiol S and k additional weak Hg···S contacts, or 2 + k coordination. The calculated stabilization energy is -5 kcal/mol per RSR ligand. Detection of distant ligands beyond the first coordination shell requires precise measurement of, and comparison to, spectra of reference compounds as well as accurate calculation of spectra for representative molecular models. The combined experimental and theoretical approaches described here for Hg can be applied to other closed-shell atoms, such as Ag(I) and Au(I). To facilitate further calculation of XANES spectra, experimental data, a new crystallographic structure of a key mercury thioether complex, Cartesian coordinates of the computed models, and examples of input files are provided as Supporting Information .