Thermodynamics of Hg(II) Bonding to Thiol Groups in Suwannee River

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Environmental Processes

Thermodynamics of Hg(II) Bonding to Thiol Groups in Suwanee River Natural Organic Matter Resolved by Competitive Ligand Exchange, Hg LIII-edge EXAFS and 1H NMR Spectroscopy Yu Song, Tao Jiang, Van Liem-Nguyen, Tobias Sparrman, Erik Björn, and Ulf Skyllberg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00919 • Publication Date (Web): 07 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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Environmental Science & Technology

Thermodynamics of Hg (II) Bonding to Thiol Groups in Suwanee River Natural Organic Matter Resolved by Competitive Ligand Exchange, Hg LIIIedge EXAFS and 1H NMR Spectroscopy

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Yu Song,† Tao Jiang,† Van Liem-Nguyen,‡§ Tobias Sparrman,‡ Erik Björn,‡ and Ulf Skyllberg*,†

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†

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SE-901 83 Umeå, Sweden

Department of Forest Ecology and Management, Swedish University of Agricultural Science,

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‡

Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden

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§

School of Science and Technology, Örebro University, SE-701 82 Örebro, Sweden

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*Corresponding author: Ulf Skyllberg. Phone: +46 (0)90-786 84 60; e-mail: [email protected]

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1 ACS Paragon Plus Environment


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ABSTRACT

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A molecular level understanding of the thermodynamics and kinetics of the chemical bonding

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between mercury, Hg(II), and natural organic matter (NOM) associated thiol functional groups

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(NOM-RSH) is required if bioavailability and transformation processes of Hg in the environment

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are to be fully understood. This study provides the thermodynamic stability of the Hg(NOM-

21

RS)2 structure using a robust method in which cysteine (Cys) served as a competing ligand to

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NOM (Suwanee River 2R101N sample) associated RSH groups. The concentration of the latter

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was quantified to be 7.5 ± 0.4 µmol g−1 NOM by Hg LIII-edge EXAFS spectroscopy. The

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Hg(Cys)2 molecule concentration in chemical equilibrium with the Hg(II)-NOM complexes was

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directly determined by HPLC-ICPMS and losses of free Cys due to secondary reactions with

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NOM was accounted for in experiments using 1H NMR spectroscopy and 13C isotope labeled

27

Cys. The log K ± SD for the formation of the Hg(NOM-RS)2 molecular structure, Hg2+ +

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2NOM-RS− = Hg(NOM-RS)2, and for the Hg(Cys)(NOM-RS) mixed complex, Hg2+ + Cys− +

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NOM-RS− = Hg(Cys)(NOM-RS), were determined to be 40.0 ± 0.2 and 38.5 ± 0.2, respectively,

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at pH 3.0. The magnitude of these constants was further confirmed by 1H NMR spectroscopy and

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the Hg(NOM-RS)2 structure was verified by Hg LIII-edge EXAFS spectroscopy. An important

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finding is that the thermodynamic stabilities of the complexes Hg(NOM-RS)2, Hg(Cys)(NOM-

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RS) and Hg(Cys)2 are very similar in magnitude at pH values < 7, when all thiol groups are

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protonated. Together with data on 15 low molecular mass (LMM) thiols, as determined by the

35

same method (Liem-Ngyuen et al., 2017),1 the constants for Hg(NOM-RS)2 and Hg(Cys)(NOM-

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RS) represent an inherently consistent thermodynamic data set which we recommend is used in

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studies where the chemical speciation of Hg(II) is determined in presence of NOM and LMM

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thiols.


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INTRODUCTION

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Complexes formed between inorganic, divalent mercury Hg(II) and natural organic matter

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(NOM) functional groups are known to be important for the chemical speciation, bioavailability

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and transformation of Hg in the environment.2,3 Being a soft Lewis acid, Hg(II) has a strong

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affinity for thiol functional groups in NOM (NOM-RSH), which largely control the chemical

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speciation of Hg(II) under oxic conditions.2 Under sulfidic conditions, inorganic sulfide species

45

compete with NOM-RSH functional groups for Hg(II). The formation of more complex Hg(II)-

46

sulfide molecules, such as nanoparticulate (nano-HgS) and crystalline metacinnabar (β-HgS),4,5

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is further influenced by secondary effects of NOM-RSH on rates of nucleation and growth of

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HgS colloids,4, 6-11 which subsequently affect bio-uptake and transformation of Hg.3, 12-15

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Passive diffusion16, 17 and active transport18 are proposed mechanisms for cellular uptake of

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small, neutral Hg(II) molecules, such as HgCl20, Hg(SH)20, while Hg(II) complexes formed with

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low molecular mass thiols (LMM-RSH) are proposed to be actively transported across cell

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membranes.18, 19 To add complexity, thiol (RSH) functionalities located in the very membrane

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(or wall) of cells will influence the bio-uptake of Hg(II),20-24 as a consequence of their

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competition with thiol groups of NOM and low molecular mass (LMM) compounds outside the

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cell.25-27 Thus, to fully understand mechanisms and rates of cellular uptake of Hg(II), information

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about concentrations of all these different types of thiols, as well as data on the thermodynamics

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and kinetics of complexes formed between the thiols and Hg(II), is required.

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Although much progress has been made the last decades, there are uncertainties regarding the

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thermodynamic stability of complexes formed between Hg(II) and NOM-RSH functional groups,

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including possible mixed ligation with LMM thiols and other soft ligands such as I−.1 There is

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compelling evidence from Hg EXAFS spectroscopy measurements of a stable Hg(NOM-RS)2 3 ACS Paragon Plus Environment


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structure.28-31 This structure is expected to be the dominant Hg(II) form in non-sulfidic

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environments, even if some doubt recently was raised about its long-term stability.31 In a critical

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review, the value of the formation constant for the structure Hg(NOM-RS)2, log KHg(NOM-RS)2, was

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constrained to 42 ± 2.2 The variability reported is expected to be mainly related to methodology,

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where constants selected for competing ligands (e.g. Br− and thiol ligands) in experimental work,

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as well as pKa values for the NOM-RSH groups are particularly critical.2 So far there are only

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two studies in which the concentration of NOM-RSH groups was quantified and a molecular

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reaction for the formation of the Hg(NOM-RS)2 structure was formulated.32,

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proposed Hg(NOM-RS)2 molecular structure has so far not been independently verified by

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spectroscopic measurements in any of the existing reports presenting experimental data on the

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stability constant log KHg(NOM-RS)2.

33

Notably, the

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Because of the exceptionally strong bonding of Hg(II) to NOM-RSH groups, thermodynamic

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experiments require addition of a potent competing ligand with known stability constants for

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Hg(II) complex formation.33-37 LMM molecules containing a thiol group have been used for this

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purpose.33, 35, 38 Liem-Nguyen and co-workers recently developed two robust mass spectrometry-

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based methods by which specific LMM-RSH molecules and their Hg(LMM-RS)2 complexes

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could be determined at sub-nM levels.1, 39

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In this work we used cysteine (Cys) as a competing ligand to determine the stability constant

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for the Hg(NOM-RS)2 structure, using the well characterized and in research frequently applied

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Suwanee River NOM (2R101N, International Humic Substances Society, IHSS). We used Hg

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LIII-edge EXAFS spectroscopy to determine the concentration of NOM-RSH functional groups

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and to verify the Hg(NOM-RS)2 structure in our experiments. Thermodynamic constants were

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derived from data on the equilibrium concentration of the Hg(Cys)2 complex, as determined by 4 ACS Paragon Plus Environment

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HPLC-ICPMS.1 Further, the presence of a mixed complex, Hg(Cys)(NOM-RS), with Hg(II)

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bonded to one Cys− and one NOM-RS− group was proposed, based on 13C-Cys isotope labelling

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experiments. We optimized the values on the thermodynamic stability constants (log K) for the

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Hg(NOM-RS)2 and Hg(Cys)(NOM-RS) structures by a least square procedure using data

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obtained at varying Hg(II) to NOM mass ratios. Thermodynamic stabilities were further

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validated by proton nuclear magnetic resonance (1H NMR) spectroscopy. The direct and

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simplistic approach, avoiding liquid-liquid and solid-phase extraction steps, as well as the

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independent, spectroscopic determination of structures and thiol groups associated with NOM is

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expected to result in a lower uncertainties in the values of reported constants, as compared to

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previous estimates.

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MATERIALS AND METHODS

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Materials. All solutions and reagents were prepared in degassed Milli-Q water (18

98

MΩ·cm) inside a N2 (g) filled glovebox (COY). Milli-Q water was deoxygenated by purging

99

with nitrogen gas overnight in the glovebox. A Hg(II) stock solution (6.5 mM) was prepared

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from Hg(NO3)2 in 1% nitric acid. The concentration of Hg(II) stock solution was verified by

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reversed isotope dilution analysis using ICPMS (Elan DRC-e) and by combustion atomic

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absorption spectrometry (C-AAS, DMA-80). Stock solutions of 0.1 mM of low molecular mass

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thiols (Sigma-Aldrich) i.e., cysteine (Cys), homocysteine (HomoCys) and N-Acetyl-L-cysteine

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(NACCys) were freshly prepared and stored in the glovebox no more than 24 h before use.

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We used Suwannee River NOM (2R101N), obtained from IHSS in this study. The

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concentration of major elements in this material is displayed in Table S1, where in particular the



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concentration of Cl should be noted. Stock solutions of NOM (~500 mg L−1) were freshly

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prepared by dissolving the NOM material in deoxygenated Milli-Q water in the glovebox. The

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stock solution was filtered through a 0.22-µm filter (Millipore) before use, and its concentration

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was confirmed by a total organic carbon (TOC) analyzer (Shimadzu TOC-V + TNM1). Losses of

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TOC were less than 2% in the filtration step, indicating a close to complete dissolution of the

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NOM material.

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Chemical Equilibrium Experiments and Thermodynamic Calculations in the Hg-

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NOM-Cysteine System. A series of NOM solutions (10, 20, 40, 80 and 200 mg L−1) were

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prepared in duplicates in 15 mL polypropylene tubes (Sarstedt), by diluting the NOM stock

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solution in deoxygenated Milli-Q water added NaClO4 to provide an inert, constant ionic

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medium of 10 mM. To these NOM solutions, aliquots of Hg(II) stock solution were added to

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obtain a final volume of 10 mL and concentration of 0.5 µM Hg(II). To avoid introducing

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potential interferences no pH-buffer was added. Because of the numerous NOM acid-base

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functional groups, pH was maintained at 3.1 ± 0.2 in all Hg-NOM experiments. Reaction vessels

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were protected from light by aluminum foil and were maintained at 25 ± 1 °C by a thermostat in

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the glove-box. The Hg–NOM solutions were gently mixed in an anaerobic glove-box for 24 h.

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Although the reaction of Hg(II) with NOM is expected to be kinetically controlled,40 Hg LIII-

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edge EXAFS data demonstrated that added Hg(II) was complexed by two NOM-RS− groups

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after 24 h, as well as after 5 days of reaction, as depicted by reaction (1):

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Hg2+ + 2NOM-RS− = Hg(NOM-RS)2

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(1)

After 24 h of reaction between Hg(II) and NOM, 2 µM of Cys was added as a competing ligand to the NOM-RS− groups, as described by reaction (2).


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Hg(NOM-RS)2 + 2Cys− = Hg(Cys)2 + 2NOM-RS−

(2)

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Here we define HCys as the cysteine molecule with the carboxyl group deprotonated (–

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COO−) and the thiol (–SH) and amino groups (–NH3+) protonated, HSCH2CH(NH3+)COO−, and

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Cys− refers to the molecule when both the carboxyl and thiol groups are deprotonated. As

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established in a pilot study, a final concentration of 2 µM of Cys was chosen to provide a

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measurable concentration of the Hg(Cys)2 complex in all experiments. Because Cys may be

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slowly oxidized by NOM, especially at high pH,41 we chose to keep a low pH in our experiments.

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Parallel experiments with 0.5 µM Hg(II) and 2 µM Cys prepared in deoxygenated Milli-Q water

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in absence of NOM, and with pH adjusted to 3.0 by nitric acid, were used as controls.

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We conducted Cys ligand-exchange experiments to determine the log KHg(NOM-RS)2 for

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reaction (1), as defined by equation (3),

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K Hg ( NOM -RS )2 =

141

in which {} denotes activity and [] denotes equilibrium concentration in mol L−1. All activities

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were calculated by the extended Debye-Hückel equation.42 Because the NOM-RS− and

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Hg(NOM-RS)2 molecules are part of a mixture of differently sized, unknown macromolecules,

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activities cannot be specified and we therefore use concentrations for these two components. The

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term {Hg2+} in equation (3) was substituted for the quotient

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the law of mass action (equation 5) for reaction (4):

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Hg2+ + 2Cys− = Hg(Cys)2,

(4)

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K Hg ( Cys )2 =

{Hg(Cys)2 } {Hg 2+ }{Cys − }2

(5)

[Hg(NOM-RS) 2 ] {Hg 2+ }[NOM-RS− ]2

(3)


{Hg(Cys)2 } as derived from K Hg (Cys )2 {Cys − }2


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yielding equation (6).

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K Hg ( NOM -RS )2

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The value of KHg(Cys)2 was set to 1037.5, following Liem-Nguyen et al.1

K Hg ( Cys )2

[Hg(NOM-RS) 2 ]{Cys − }2 = {Hg(Cys) 2 }[NOM-RS− ]2

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(6)

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Of the four unknowns on the right side of equation (6), the concentration of the Hg(Cys)2

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complex was directly determined by HPLC-ICPMS (see below) and {Cys−} was calculated from

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[Cys−] by difference using the mass balance equation (7).

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[Cysadded] = [Cys−] + [HCys] + 2[Hg(Cys)2] + [Cys–NOM] + [Hg(Cys)(NOM-RS)]

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The term Cys–NOM represents Cys which is reacted with NOM components, including losses

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due to degradation, as quantified in specific experiments (see below). The Hg(Cys)(NOM-RS)

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structure is a proposed complex formed by a mixture of one Cys− and one NOM-RS− functional

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group, as described by reaction (8)

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Hg2+ + Cys− + NOM-RS−= Hg(Cys)(NOM-RS)

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The concentration of the mixed complex was estimated based on experiments in which

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labeled Cys was allowed to react with NOM in absence and presence of Hg(II), as described in

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Supporting Information, and by a final refinement of the chemical speciation model (Table S2).

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Concentrations of HCys and Cys− were calculated using a Ka value of 10−8.6 for reaction (9).1

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CysH = Cys− + H+, Ka = 10−8.6

(7)

(8) 13

C

(9)

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Finally, to solve the two remaining unknowns in equation (6), Hg(NOM-RS)2 and NOM-

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RS−, the concentration of Hg(NOM-RS)2 was calculated from the mass balance equation (10).

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The total Hg concentration [Hgtot] was measured by C-AAS. Other Hg(II) components, like the 8 ACS Paragon Plus Environment

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free Hg2+ ion and its complexes with OH− and Cl− (reactions which for completeness are

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included in the chemical speciation model, Table S2) all made negligible contributions to the

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mass balance.

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[Hgtot] = [Hg(Cys)2] + [Hg(NOM-RS)2] + [Hg(Cys)(NOM-RS)]

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The total concentration of NOM-associated thiol groups [NOM-RStot] was determined by a Hg2+

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titration procedure of these groups monitored by Hg LIII-edge EXAFS, as described below. The

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[NOM-RS−] component in equation (6) was calculated from the mass balance (11) and the pKa

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value of reaction (12) was set to 10.0, following Skyllberg.2

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[NOM-RStot] = [NOM-RS−] + [NOM-RSH] + 2[Hg(NOM-RS)2] + [Hg(Cys)(NOM-RS)] (11)

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NOM-RSH = NOM-RS− + H+, Ka = 10−10.0

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All reactions and selected thermodynamic constants considered in our model are listed in Table

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S2. It should be noted that because of the exceptionally strong covalent bond formation between

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Hg(II) and NOM-RS−, electrostatic forces involving negatively charged NOM functional groups

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is expected to have insignificant impact on the chemical speciation. Calculations were conducted

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using the chemical speciation computer programs PHREEQC43 and WinSGW.44 Errors in the

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reported values of KHg(NOM-RS)2 and KHg(Cys)(NOM-RS) were propagated by use of equation (13),

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where SDi denotes the standard deviation of error source i.

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SD =

∑ SD

(10)

(12)

2 i

(13)

i

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Spectroscopic determinations. The sulfur K-edge XANES, Hg LIII-edge EXAFS and 1H

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NMR spectroscopy methods were used to determine the concentration of NOM-RStot functional

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groups, to identify structures of Hg(II)-NOM complexes, and to provide independent 9 ACS Paragon Plus Environment


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thermodynamic data. The concentration of the Hg(Cys)2 complex was determined by HPLC-

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ICPMS according to Liem-Nguyen et al.1 A description of these methods is given in the

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Supporting Information. The concentration of Cys was determined by LC-ESI-MS/MS, using a

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procedure described previously.39 Finally, we used 13C isotope labeled Cys (13C-Cys) to monitor

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possible degradation of Cys and reaction with NOM functional groups in experiments, as

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described in the Supporting Information.

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RESULTS AND DISCUSSION

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Hg LIII-Edge EXAFS Determinations of the Concentration of NOM-RStot. We used a Hg(II)

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titration procedure, monitored by Hg LIII-edge EXAFS, to determine the concentration of thiol

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functional groups in NOM, NOM-RStot. The NOM samples were allowed to react with 800, 1600

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and 3300 µg of Hg(II) per gram of dry mass of NOM for 5 days in the glove-box. After freeze-

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drying, pelleting, and storage at −80 oC and EXAFS measurements, the measured Hgtot

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concentrations were determined to be 780, 1400 and 2650 µg g−1, respectively. Some loss of Hg

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in experiments with NOM is expected to occur as a consequence of the reduction of Hg(II) to

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Hg(0) by NOM under dark conditions,40,

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experiments of this study (see below).

45, 46

which was also observed in the equilibrium

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In agreement with previous research,28 EXAFS determinations revealed a Hg(NOM-RS)2

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structure formed between Hg(II) and two thiol groups in NOM after 5 days of reaction. At the

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lowest Hg(II) concentration of 780 µg g−1, an average Hg–S bond distance of 2.34 Å was

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obtained when the coordination number (CN) was fixed to 2.0. The result provides strong

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support for the Hg(NOM-RS)2 structure.28,47 We did not add any chloride, but the sample


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contained Cl− (Table S1) due to the method used by IHSS to extract and up-concentrate the

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Suwanee River NOM sample. In the Hg(II)-NOM suspensions prepared for EXAFS

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determinations, in which a minimum of water was added to improve signal-to-noise ratio, we

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measured 6 mM of Cl− ions. Since Cl− ions are known to form bonds with Hg(II) that are

216

stronger than associations with oxygen functionalities in NOM, we included a Hg–Cl bond in our

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model and fixed it to 2.28 Å in agreement with the HgCl2 complex.48, 49 To constrain Cl and S

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bonding to Hg(II) in a similar way, we also fixed the Hg–S bond distance (at 2.35 Å) in our final

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modelling, which is a typical distance of the Hg–S bond in a Hg(SR)2 structure.

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The first coordination shell model fits to full EXAFS k-space data are reported in Table 1

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and fits to FT back-filtered data are reported in Table S3. As expected, the coordination number

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of Cl increased with increasing addition of Hg(II) (Figure 1, Table 1). Thus, when the RS groups

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in NOM were increasingly saturated by Hg(II), the coordination with the more weakly bonded

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ligand Cl− increased. Inclusion of a 1st shell Hg–Cl bond at 2.28 Å significantly improved the

225

model fits to FT back-filtered 1st shell data for samples added 780, 1400 and 2650 µg g−1 Hg(II),

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by 43, 90, and 94% respectively. Although the Hg–Cl bond distance in two-coordinated Hg(II)

227

complexes is only ~0.07 Å shorter than the Hg–S bond length, the extraordinary good signal-to-

228

noise ratio in our data enabled us to separate the contribution from Hg–S and Hg–Cl bonding.

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Because of the increased contribution of Cl (and decrease in the contribution of S), the radial

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distance of the 1st coordination shell peak decreased slightly with Hg(II) loading (Figure 1b).

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In the second coordination shell, inclusion of one S atom at 2.95 Å and two Cl atoms at

232

~3.3 and ~3.5 Å, respectively, significantly improved model fits by > 20% (Table S4). The 2nd

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shell S distance is in agreement with previous studies of Hg(II)-NOM complexation,28,47

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reporting an organic sulfide contribution at 2.95–3.05 Å, while the 2nd shell Cl distances are in 11 ACS Paragon Plus Environment


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agreement with the crystalline structure of HgCl2(s).49 Possibly the freeze-drying of samples

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gave rise to some formation of HgCl2(s) crystals when water was removed, alternatively the

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structure observed in the 2nd shell reflects a contribution from dissolved Cl− ions, combining with

238

Hg(NOM-RS)2 and aqueous HgCl2, and/or possible mixed HgCl(NOM-RS) structures. In either

239

case, the inclusion of Cl in 1st and 2nd shells improved the merit-of-fit for the full k-space data by

240

22, 70 and 51% for NOM samples added 780, 1400 and 2650 µg g−1 Hg(II), respectively.

241

Based on the Hg EXAFS data, the concentration of thiol groups (NOM-RStot) in the

242

Suwanee River NOM sample could be calculated by equation (14). The assumption behind this

243

equation is that all thiol groups take part in the bonding of Hg(II) before significant numbers of

244

Cl− ligands get involved.

245

[NOM-RStot ] =

246

In this equation, CNS and CNCl denote the 1st shell coordination numbers of S and Cl atoms,

247

respectively, and [] denotes molar concentrations per mass of NOM. Using the model fits to full

248

EXAFS k-space data, the concentration of NOM-RStot was calculated to be 7.0, 7.5 and 7.9 µmol

249

g−1 for samples with Hg(II) concentrations of 780, 1400 and 2650 µg g−1, respectively. Thus we

250

obtained very similar estimates of the NOM-RStot concentration in all three samples, suggesting

251

our model assumptions were reasonable. As the final estimate of the NOM-RStot, we used the

252

average value of 7.5 ± 0.4 µmol g−1 (NOM). To further strengthen our model assumptions, we

253

conducted theoretical thermodynamic calculations (using the data in Table S2). Using the

254

average NOM-RStot concentration of 7.5 µmol g−1, the calculated relative abundance of the two

255

major structures Hg(SR-NOM)2 (97, 54 and 28%) and HgCl2 (3, 45 and 69%, Table S5) was

CN S × 2[Hg] CN S + CN Cl

(14)


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well in agreement with the EXAFS data (90, 54 and 29% for Hg(SR-NOM)2 and 10, 46 and 71%

257

for HgCl2), within limits of uncertainty of both methods (Table 1, Table S5).

258

It should be pointed out that also carboxylic and phenolic groups in NOM may form

259

complexes with Hg(II) when NOM-RS groups are saturated.40 However, because these

260

associations are substantially weaker than Hg–Cl bonds we could not observe any O/N atoms in

261

the 1st coordination shell of our EXAFS data. This is reasonable given that the sum of the

262

concentration of NOM-RStot and Cl− were always in much excess of Hg(II) (considering its 2+

263

charge) in the experimental systems. The insignificant contribution from O/N containing

264

functional groups of NOM in the bonding of Hg(II) was also predicted by the thermodynamic

265

modelling (Table S5).

266

By use of S K-edge XANES we determined the concentration of reduced organic sulfur

267

functionalities (Org-SRED, representing the sum thiol RSH, monosulfide RSR and disulfide

268

RSSR)32 to account for 10% of total sulfur (Figure S2). This is a small percentage of Org-SRED as

269

compared to previous studies of NOM samples,50,

270

concentration of inorganic sulfate in the Suwanee River NOM sample. Similar to Cl−, sulfate

271

ions are up-concentrated in the NOM sample due to the IHSS extraction procedure (Table S1).

272

Recalculated to mass of NOM, Org-SRED makes up 56 µmol g−1 of NOM which is at the same

273

level as in previous studies of similar types of NOM samples.2,28 A comparison with the Hg LIII-

274

edge EXAFS determined concentration of 7.5 µmol g−1 of NOM-RStot suggests these groups

275

accounted for 15% of Org-SRED, which is a little lower than the average of previous studies of

276

NOM from soils and waters, but still within the expected range of 10–35%.2, 28, 50, 52 Notably, no

277

forms of inorganic sulfide, such as FeS(s) or nano-particulate HgS, were detected by neither S K-

278

edge XANES nor by the Hg LIII-edge EXAFS analyses.

51

which can explained by the high



279

Hg-NOM-Cysteine Ligand-Exchange Experiments. We conducted ligand-exchange

280

experiments, allowing Cys to compete with NOM-RS functional groups for the complexation of

281

Hg(II). Prior to the addition of Cys , Hg(II) was reacted with NOM for 24 h to form the

282

Hg(NOM-RS)2 structure. The dominance of this structure was independently demonstrated by

283

use of Hg LIII-edge EXAFS for a NOM sample added Hg(II) corresponding to 800 µg g−1. The

284

first coordination shell Hg–S distance was 2.36 Å and the coordination number (CN) 2.0 (data

285

not shown). Because the values of the EXAFS parameters (R, CN, σ2) did not differ from the

286

ones obtained after 5 days of Hg(II)-NOM reaction (Table 1), we suggest the system reached

287

close to a chemical equilibrium within 24 h of reaction. Notably, because this EXAFS sample

288

was prepared in only 1 mL of Milli-Q (see Supporting Information) the concentration of Cl− was

289

estimated to be 6 mM and the concentration of NOM-RStot would be expected to have been close

290

to saturated by the added Hg(II). Yet, inclusion of Cl as a 1st shell backscatter did not

291

significantly improve model fits.

292

After addition of Cys, the concentration of the Hg(Cys)2 molecule was directly measured

293

at different times of reaction by HPLC-ICPMS. After a rapid reaction and increase within

294

minutes, the concentration of Hg(Cys)2 remained constant (within 10% RSD) in the time frame

295

from 5 min to 5 d (Figure S3). It should be noted that the concentration of Cl− was below 0.15

296

mM in all Hg-NOM-Cys ligand-exchange experiments and the formation of Hg-Cln2-n complexes

297

therefore can be ruled out as being of importance for the results.

298

After the addition of Cys, Hg EXAFS data collected at a Hg/NOM mass ratio of 800 µg

299

g−1 and at a Cl− concentration less than 0.15 mM demonstrated a 1st shell Hg(II) coordination

300

with ~2 S atoms at a distance of 2.33–2.34 Å (Figure S4, Table S6), which did not change with

301

time of reaction (1–120 h). The EXAFS data can be understood from the HPLC-ICPMS 14 ACS Paragon Plus Environment

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measurements conducted in experiments with a similar Hg/NOM ratio (0.5 µM of Hg reacted

303

with 200 mg NOM L−1, corresponding to 500 µg g−1, and then added 2 µM Cys). As seen in the

304

Figure S3e, ~50% of Hg(II) was in the form of the Hg(Cys)2 complex (~0.2 µM) and the

305

remaining ~50% was bonded with NOM, obviously in the Hg(NOM-RS)2 structure, as

306

demonstrated by EXAFS and thermodynamic modelling (see below). From Hg EXAFS

307

determinations alone we cannot distinguish if the Hg(NOM-RS)2 and Hg(Cys)2 complexes only

308

exist in these distinct forms or if they partly combine in a mixed Hg(Cys)(NOM-RS) complex.

309

This is because Hg(II) is bonded with two thiol groups at 2.33–2.36 Å in all three structures.

310

Even if we cannot prove chemical equilibrium by reversibility in the Hg-NOM-Cys

311

experimental system, the fact that the 1st coordination shell (as determined by EXAFS) remained

312

the same at 1 h, 24 h and 5 days of reaction after Cys addition (Figure S4, Table S6), and that the

313

measured Hg(Cys)2 concentration (as determined by HPLC-ICPMS), once quickly formed, did

314

not change with time (Figure S3) indicate that chemical equilibrium was achieved among

315

Hg(Cys)2, Hg(NOM-RS)2 and Hg(Cys)(NOM-RS) complexes already within minutes of reaction.

316

This interpretation is further supported by the thermodynamic modeling (see below).

317

Notably the measured Hgtot concentrations were 20–40% lower in the Hg-NOM-Cys

318

systems than what was expected from the additions, and losses varied with the Hg/NOM ratio

319

(Figure 2 and Figure S1). All losses occurred during the first 24 h of reaction between Hg(II) and

320

NOM in absence of Cys. In control systems without NOM there was no Hg loss (Figure S1).

321

This observation is in agreement with previous findings that Hg(II) is reduced to Hg(0) in

322

presence of NOM under dark conditions.45, 46 Losses are greatest during the first minutes to an

323

hour of reaction when equilibrium is not established, when weaker NOM bonds are expected to

324

be involved in the complexation of Hg(II).40 Since the reaction tubes were sealed during reaction, 15 ACS Paragon Plus Environment


325

losses likely occurred as Hg(0) evasion when they were opened for Hgtot analyses, or possibly

326

formed Hg(0) could have been sorbed to the walls of the tubes.

327

To further deepen our understanding of the kinetics of Hg(II) bonding to thiol groups

328

belonging to different molecules, the reaction between N-Acetyl-L-cysteine (NACCys),

329

homocysteine (HomoCys) and Hg(II) was experimentally studied (see Supporting Information).

330

Concentrations of Hg(HomoCys)2 and Hg(NACCys)2 were demonstrated to reach equilibrium

331

within less than 3 minutes (Figure S5). This observation is in agreement with previous studies

332

showing that although the Hg(II)–thiol bond is very strong, the rate of ligand-exchange among

333

Hg(II)–thiol complexes is on the order of seconds.53 The latter study demonstrated Hg(II)

334

complex formation with a mixture of thiol groups pertaining to a relatively small, glutathione

335

(GSH) and large molecules, hemoglobin (having eight thiol groups). These heterogenic Hg(II)-

336

thiol complexes were shown by 1H NMR spectroscopy to have lifetimes of less than 30 s due to

337

quick ligand-exchange among GSH molecules. Similarly, it seems as rates of reactions between

338

Cys and large macromolecules, including NOM-RS functional groups, also are very quick, at

339

least when the initial condition is dominated by the Hg(NOM-RS)2 structure.

340

Reaction Between Cys and NOM and Formation of a Mixed Hg(Cys)(NOM-RS)

341

Complex. LMM-RS compounds are known to react with RSH functional groups under the

342

formation of organic disulfides,54 and because Cys is sensitive to degradation in presence of

343

NOM, e.g. through oxidation,41 it was important to monitor the concentration of free Cys over

344

time in our experiments. As shown by LC-ESI-MS/MS determinations (Figure S6), Cys was not

345

degraded in the absence of NOM. Similar results were obtained by 1H NMR, with less than 5%

346

of Cys degraded after 5 days (Figure S7a).


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347

However, in the presence of NOM (1000 mg L−1) the concentration of free Cys (100 µM)

348

decreased by 30% in 5 days (Figure S7b), suggesting reactions taking place between Cys and

349

NOM. Similarly,

350

Information for details on how

351

after 5 days (Figure 3). The ~ 10 times higher Cys to NOM ratio may be the reason for slightly

352

higher reactivity of Cys with NOM in the 1H NMR experiment.

353

13

C-Cys reacting with NOM showed a time-dependency (see Supporting 13

C data were interpreted) increasing from 1% after 1 h to 25%

As compared to reaction with NOM alone,

13

C-Cys reacted with NOM increased by in

354

average 7% if NOM was allowed to react with Hg(II) for 24 h before Cys was added. This

355

increase in NOM-associated Cys is illustrated by the blue symbols and dashed line in Figure 3.

356

We interpret this “extra” NOM-reacted Cys in presence of Hg(II) as reflecting a formation of a

357

mixed complex with the proposed composition Hg(Cys)(NOM-RS). EXAFS data further

358

confirmed that all Hg (within an uncertainty of ± 5–10%) was indeed bonded to two thiol groups.

359

But again, the three complexes Hg(Cys)2, Hg(NOM-RS)2 and Hg(Cys)(NOM-RS) cannot be

360

separated by Hg EXAFS.

361

Thermodynamic calculations. From equations (6)–(12) the thermodynamic constant for the

362

formation of the Hg(NOM-SR)2 complex was first calculated disregarding the existence of the

363

proposed mixed Hg(Cys)(NOM-RS) complex. In a second step, we calculated the concentration

364

of the mixed Hg(Cys)(NOM-RS) structure as the difference between free 13C-Cys concentrations

365

measured in systems with and without Hg(II) addition (Figure 3), by also including equation (15).

366

K Hg (Cys )( NOM -RS ) =

[Hg(Cys)(NOM-RS)] {Hg 2+ }{Cys − }[NOM-RS− ]

(15)



367

The calculation was done by an iterative procedure where the error-sum-of-squares was

368

minimized by adjusting the two parameters KHg(NOM-RS)2 and KHg(CyS)(NOM-RS) while keeping the

369

quotient between them (= 37) the same as determined by the 13C-Cys experiment.

370

The log KHg(NOM-RS)2 was calculated to be 40.4 ± 0.2, when the mixed Hg(Cys)(NOM-RS)

371

was disregarded, and 40.0 ± 0.2 in presence of this mixed complex. In the latter case log

372

KHg(Cys)(NOM-RS) was calculated to be 38.5 ± 0.2. Fits to data of the two alternative models are

373

indicated by the dashed lines in Figures 2a and b. The sources of uncertainties propagated by

374

equation (13) for the log constants (± 0.2) are listed in Table S8. Because the relative error of the

375

fits did not differ between the two alternative models (0.02 and 0.06 %, respectively, Figure 2),

376

evidence for a mixed complex relies entirely on the observations from 13C-Cys experiments, and

377

not on the thermodynamic model fitting.

378

We used 1H NMR spectroscopy measurements to validate the thermodynamic models.

379

We determined the equilibrium concentrations of free Cys and Hg(Cys)2, Figure S8. The Hg(II)

380

concentration was required to be much higher than in experiments using HPLC-ICPMS, but the

381

Hg(II) ratio to NOM (3 000 µg g−1) was kept similar to experiments in which 0.5 µM Hg(II) was

382

added to 20 or 40 mg L−1 of NOM. The measured concentrations of Hgtot, Cys and Hg(Cys)2 are

383

tabulated together with the theoretical concentrations calculated from our final model, with log

384

KHg(NOM-RS)2 and log KHg(Cys)(NOM-RS) set to 40.0 and 38.5, respectively, in Table S7. As further

385

illustrated in Figure S9, we obtained a good correspondence between measured and model

386

predicted concentrations of Hg(Cys)2 and free Cys by use of 1H NMR.

387

We consider our reported thermodynamic stability for the Hg(NOM-SR)2 structure to be

388

the most robust reported so far. The direct determination of the Hg(Cys)2 complex formed with

389

the competing ligand, the continuous monitoring and correction for losses of Cys and the 18 ACS Paragon Plus Environment

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390

independent spectroscopic determination and verification of the concentration of NOM-RStot

391

groups and the Hg(NOM-SR)2 structure, respectively, are all major improvements as compared

392

to previous laboratory studies on Hg(II)-NOM complexation.2,32,34,33, 35, 38 In none of the previous

393

studies have the modelled structure of the Hg(II)-NOM complex been spectroscopically verified.

394

As described by Black and coworkers,34 the CLE-SPE methodology employed in several of these

395

studies to separate Hg(II)-NOM complexes from Hg(II) complexes formed with competing

396

ligands, is subjected to operational difficulties related to the dynamic character of hydrophilic

397

and hydrophobic fractions of NOM and how these fractions may be affected by and interact with

398

the competing LMM thiol ligands and the C-18 column employed for separation. With the

399

simplistic and direct approach used here, operational constraints are kept at a minimum.

400

Environmental implications. Although NOM is known to significantly influence speciation

401

and bioavailability of Hg(II), the details on the thermodynamics and kinetics of Hg(II) structures

402

formed with NOM functionalities need to be sharpened. In this study the structure and

403

thermodynamic stability of the Hg(NOM-RS)2 complex was verified by Hg LIII-edge EXAFS. It

404

should be noted that NOM-RSH is a representation of all thiol groups in NOM and that the pKa

405

and log KHg(NOM-RS)2 therefore can be considered average constants. The size of the constant for

406

Hg(NOM-RS)2 in essence confirms the magnitude of critically reviewed constants for this

407

chemical structure in NOM.2 It should, however, be noted that our reported values on the

408

thermodynamic constants (similar to other ligand-exchange studies) heavily depends on the pKa

409

value of the competing ligand (HCys) and on the log K value of the formation of Hg(Cys)2. For

410

every log-unit increase in log KHg(Cys)2 the log K for the formation of Hg(NOM-RS)2 and

411

Hg(Cys)(NOM-RS) will increase by one log-unit and for every log-unit increase in the pKa for

412

HCys the KHg(NOM-RS)2 and KHg(CyS)(NOM-RS) will increase by two and one log-units, respectively.



Page 20 of 31

413

At acidic and neutral pH conditions, the thiol groups of HCys and NOM-RSH will be protonated

414

and the relative stabilities of Hg(Cys)2, Hg(NOM-RS)2 and Hg(Cys)(NOM-RS) can be described

415

by a general reaction (16), where RSH denotes a thiol group on either Cys or NOM:

416 417

Hg2+ + 2RSH = Hg(RS)2 + 2H+, log KHg(RS)2

(16)

418 419

Formulated in agreement with reaction (16) the log KHg(Cys)2, KHg(NOM-RS)2 and KHg(CyS)(NOM-RS)

420

have the values 20.3, 20.0 and 19.9, respectively (calculated as 37.5 − 2 × 8.6, 40.0 − 2 × 10.0

421

and 38.5 − 8.6 − 10.0, respectively). This means that all three complexes in essence (within

422

experimental errors of ± 0.2) have equal thermodynamic stabilities at pH-values when the thiol

423

groups are protonated. This is a very important finding, since it tells us that regardless of values

424

chosen for the pKa of HCys and log K for Hg(Cys)2, which vary substantially in the literature, we

425

may adjust constants to be consistent with our finding that Hg(Cys)2, Hg(NOM-RS)2 and

426

Hg(Cys)(NOM-RS) have equal stabilities at and below neutral pH. This would be particularly

427

important in systems where thiol compounds compete and control the chemical speciation and

428

bioavailability of Hg(II), such as at surfaces of bacteria.

429

To extend this reasoning further, we argue for the usage of internally consistent

430

thermodynamic models. Using the same HPLC-ICPMS methodology as in this work, Liem-

431

Nguyen et al.1 reported log KHg(RS)2 values for 15 different LMM thiols. Because Cys was part of

432

that study, it means that this entire set of constants for LMM thiols is consistent with the values

433

on log KHg(NOM-RS)2 and KHg(CyS)(NOM-RS) reported in this work. We therefore recommend using the

434

stability constants for Hg(II) complex formation with LMM thiols from the study of Liem-


Page 21 of 31


435

Nguyen et al.1 in combination with the thermodynamic constants reported for Hg(NOM-RS)2

436

and Hg(Cys)(NOM-RS) as reported here.

437

In our experiments, concentrations of Cys (2 µM) were about 10–1000 times higher than

438

normally encountered in soils and waters. Yet, the indicated large stability of the Hg(Cys)(NOM-

439

RS) complex and the assumption that other LMM thiols may also form similar mixed complexes

440

with NOM-RS suggest these complexes may be important for transportation and bio-uptake of

441

Hg(II), e.g. by methylating bacteria. A simple calculation, in which concentrations of Hg(II) (1–

442

10 ng L−1), NOM (5–100 mg L−1), Cys (1–100 nM), pH 3–7 were varied to cover typical ranges

443

in soils and waters, demonstrates that the Hg(Cys)(NOM-RS) complex can be expected to

444

generally be more abundant than Hg(Cys)2, and in cases even more abundant than Hg(NOM-

445

RS)2 (Figure S10). Future work is needed to directly confirm the structure of Hg(Cys)(NOM-RS)

446

and its bioavailability.

447

The previously proposed HgNOM-RS+ (Hg2+ + RS− = HgRS+) species55,

56

was not

448

detected by our EXAFS measurements. Since one-coordinated Hg(II) complexes are not

449

thermodynamically stable57 a more correct structure of the HgSR+ species is a two-coordinated

450

complex with one thiol and one oxygen (or nitrogen) functionality, Hg(NOM-RORS), where the

451

mathematic formulation suggests the two functional groups are belonging to the same molecule

452

(forming a bidentate complex). According to the linear free energy relationships reported by

453

Dyrssen & Wedborg56 the log K for such a HgRS+ molecule should be ~22. Our thermodynamic

454

modelling constrained the log K for such a complex to be smaller than ~26.

455

Neither did our EXAFS determinations on Hg-NOM-Cys systems revealed any sign of

456

O/N functionalities, indicative of a formation of the HgCys+ or Hg(NOM-RS)+complex, where

457

Hg(II) forms a bidentate complex with a combination of thiol and carboxyl functionalities of a



458

single Cys or NOM molecule. Given ± 5% detection limit of possible O/N functionalities by

459

EXAFS we estimate the log K for the HgCys+ species to be smaller than ~26. Our HPLC-ICPMS

460

measurements further constrained the log K to a maximum of ~24. Within methods uncertainties

461

these values are in fair agreement with data from Liem-Nguyen et al.1 Notably, their reported log

462

K value of 30.0–31.5 for HgRS+ complexes formed with a variety of LMM thiols was incorrectly

463

expressed as monodentate complexes in which Hg(II) was complexed by separate carboxyl and

464

thiol ligands, Hg(IRO)(IIRS). A correct modeling of the same experimental data with Hg(II)

465

complexed by carboxyl and thiol groups of the same molecule, Hg(RORS), results in a log K

466

value for HgCys+ of ~25 ± 0.5 (for details, see Supporting Information).

467

468

ASSOCIATED CONTENT

469

Supporting Information

470

The supporting information is available free of charge on the ACS Publications website. SI

471

contains Text, 8 Tables and 10 Figures.

472 473

AUTHOR INFORMATION

474

Corresponding Author

475

*Phone:+46-90-786 8460; email: [email protected]

476 477

ACKNOWLEDGMENTS 22 ACS Paragon Plus Environment

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478

This work was financially supported by the Swedish Research Council (VR) project Sino-

479

Swedish Mercury Management Research Framework – SMaReF (2013-6978) and the project

480

(621-2014-5370) to US, and by the Kempe Foundations (JCK-1501, SMK-2745, SMK-1243).

481

We acknowledge Dr. Roberto Boada and Ann-Kathrin Geiger at the Diamond Light Source

482

(Beamline I20-scanning), for assistance with the Hg LIII-edge EXAFS spectroscopy

483

measurements. We also acknowledge Dr. Chenyan Ma at the Beijing Synchrotron Facility

484

(Beamline 4B7A), Chinese Academy of Sciences, for assistance with the S K-edge XANES

485

spectroscopy measurements.

486

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49. Boada, R.; Cibin, G.; Coleman, F.; Diaz-Moreno, S.; Gianolio, D.; Hardacre, C.; Hayama, S.; Holbrey, J. D.; Ramli, R.; Seddon, K. R.; Srinivasan, G.; Swadzba-Kwasny, M., Mercury capture on a supported chlorocuprate(ii) ionic liquid adsorbent studied using operando synchrotron X-ray absorption spectroscopy. Dalton Trans 2016, 45, (47), 18946-18953. 50. Qian, J.; Skyllberg, U.; Frech, W.; Bleam, W. F.; Bloom, P. R.; Petit, P. E., Bonding of methyl mercury to reduced sulfur groups in soil and stream organic matter as determined by X-ray absorption spectroscopy and binding affinity studies. Geochim. Cosmochim. Acta 2002, 66, (22), 3873-3885. 51. Skyllberg, U.; Qian, J.; Frech, W.; Xia, K.; Bleam, W. F., Distribution of mercury, methyl mercury and organic sulphur species in soil, soil solution and stream of a boreal forest catchment. Biogeochemistry 2003, 64, (1), 53-76. 52. Liem-Nguyen, V.; Skyllberg, U.; Bjorn, E., Thermodynamic Modeling of the Solubility and Chemical Speciation of Mercury and Methylmercury Driven by Organic Thiols and Micromolar Sulfide Concentrations in Boreal Wetland Soils. Environ. Sci. Technol. 2017, 51, (7), 3678-3686. 53. Rabenstein, D. L.; Isab, A. A., A proton nuclear magnetic resonance study of the interaction of mercury with intact human erythrocytes. Biochim. Biophys. Acta 1982, 721, (4), 374-84. 54. Rabenstein, D. L.; Millis, K. K., Nuclear magnetic resonance study of the thioltransferasecatalyzed glutathione/glutathione disulfide interchange reaction. Biochim. Biophys. Acta 1995, 1249, (1), 29-36. 55. Dong, W.; Bian, Y.; Liang, L.; Gu, B., Binding constants of mercury and dissolved organic matter determined by a modified ion exchange technique. Environmental science & technology 2011, 45, (8), 3576-3583. 56. Dyrssen, D.; Wedborg, M., The sulphur-mercury(II) system in natural waters. Water, Air, & Soil Pollution 1991, 56, (1), 507-519. 57. Watts, J.; Howell, E.; Merle, J. K., Theoretical studies of complexes between Hg (II) ions and l‐ cysteinate amino acids. Int. J. Quantum Chem 2014, 114, (5), 333-339.

634


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Tables and Figures

Table 1. Least-squares 1st coordination shell model fits to full k-space Hg LIII-edge EXAFS data for NOM samples added different concentrations of Hg(II). Second coordination shell fits and merit-of-fit of full model are reported in Table S4. 1st shell S

Hg

a

1st shell Cl

Species compositiona

NOM-RStot concentrationb

Hg(NOM-RS)2 HgCl2

(µmol g−1)

(µg g−1)

ΔE0 (eV)

780

8.7

1.55 2.35(f) 0.0032(f)

0.18 2.28(f) 0.0048(f)

90%

10%

7.0

1 400

7.4

0.83 2.35(f) 0.0032(f)

0.72 2.28(f) 0.0048(f)

54%

46%

7.5

2 650

8.6

0.60 2.35(f) 0.0032(f)

1.45 2.28(f) 0.0048(f)

29%

71%

7.9

CN

R (Å)

σ2 (Å2)

CN

R (Å)

σ2 (Å2)

Calculated as CNS/(CNS + CNCl) × 100. b Calculated as CNS/(CNS + CNCl) × 2[Hg], where [Hg] is concentration in µmol g−1 NOM. ΔE0 = edge-

energy shift, CN = coordination number, R = bond distance, σ2 = Debye-Waller factor. (f) values were fixed during fitting.



a

b

Page 28 of 31

S,Cl S Cl Cl

c

2650 µg g-1

2650 µg g-1 χ(k)k3

1400 µg

g-1

Fourier Transformations

χ(k)k3

2650 µg g-1

1400 µg g-1

1400 µg g-1 780 µg g-1

780 µg g-1

780 µg g-1

2

4

6

8 k (Å-1)

10

12

14

0

2

4

Radial distance (Å)

6

2

4

6

8 k (Å-1)

10

12

14

Figure 1. Experimental Hg LIII-edge EXAFS data (thin, black solid line) and model fits (dashed lines) collected at 77 K on freeze-dried NOM samples equilibrated with Hg(II) for 5 days at pH 3 in 6 mM Cl. The blue dashed line denotes a model with only one floating 1st S shell and the red dashed line denotes the full model, including 1st, and 2nd S and Cl shells for. (a) EXAFS spectra in k-space, (b) Fourier transformed (FT) spectra not corrected for phase shift, with full model fit (in dashed red), and (c) Back-filtered FT spectra for the range 1.2-2.7 Å. In figure b vertical dash lines indicate 1st S+Cl, and 2nd S and Cl shells. Model fits to 1st shell data are given in Table 1 and model fits to 2nd shell data are given in Table S4. 2 ACS Paragon Plus Environment

Page 29 of 31


Hgtot

Hg Concentration (M)

0.5

Hg(Cys)2

Hg(NOM-RS)2

Hg(Cys)2

a

Hg(NOM-RS)2

Hg(Cys)(NOM-RS)

b

0.4 0.3 0.2 0.1 0.0 0

40

80

120

160

1

200

240

0

40

NOM (mg L )

80

120

160

1

200

240

NOM (mg L )

Figure 2. Measured concentrations (± SD) of Hg(Cys)2 and total concentrations of Hg (Hgtot) plotted as a function of NOM concentration. Experiments were conducted at pH = 3 and I = 10 mM NaClO4 by pre-equilibrating 0.5 µM Hg with NOM (10–200 mg L−1) for 24 h followed by a reaction with 2 µM Cys for 5 min to 5 days (Figure S3). Dashed lines show model fits. In (a) only the Hg(NOM-RS)2 complex was considered and the log KHg(NOM-RS)2 was determined to be 40.4 ± 0.2. The merit-of-fit, 100(∑[model-exp]2/∑[exp]2) for Hg(Cys)2 was 0.02%. In (b) also the mixed Hg(Cys)(NOM-RS) complex was included in the model and log KHg(NOM-RS)2 and log KHg(Cys)(NOM-RS) were determined to be 40.0 ± 0.2 and 38.5 ± 0.2, respectively. The merit-of-fit for Hg(Cys)2, was 0.06%.



Page 30 of 31

50 13

C-CysNOM in absence of Hg(II)

13

C-CysNOM in presence of Hg(II)

0.8

40

13

Hg( C-Cys)(NOM-RS) 0.6

30

0.4

20

0.2

10

0.0

0 0

24

48

72

96

NOM associated 13C-Cys (%)

NOM associated 13C-Cys (M)

1.0

120

Time (h) Figure 3. Concentration of

13

C-Cys associated with NOM in absence (black) and presence (red) of Hg(II), as a function of time of

reaction. Black symbols and line denote experiments where 2 µM 13C-Cys was added to 200 mg L−1 of NOM. Red symbols and line denote experiments where 0.5 µM Hg(II) was pre-equilibrated with 200 mg L−1 of NOM solution for 24 h, and then 2 µM 13C-Cys was added. Blue symbols and dashed line designate the difference of the two experiments, and is interpreted as formation of the mixed ligand complex Hg(13C-Cys)(NOM-RS). Error bars represent ± SD. The experiments were conducted at pH = 3 and I = 10 mM of NaClO4.


Page 31 of 31


Table of contents (TOC)

Total Hg

0.5

Hg Concentration (M)

(log K = 37.5)

Cys

0.4

2.35 Å

Hg

2.35 Å

Cys

(log K = 40.0)

S

0.3 0.2

2.35 Å

Hg

2.35 Å

R

S R

0.1

R

0.0 0

40

Cys

80 120 160 200 240

NOM (mg L1)

S

2.35 Å

Hg

2.35 Å

Cys


Thermodynamics of Hg(II) Bonding to Thiol Groups in Suwannee River

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