A stepwise reduction approach reveals mercury competitive binding

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Environmental Processes

A stepwise reduction approach reveals mercury competitive binding and exchange reactions within natural organic matter and mixed organic ligands Xujun Liang, Xia Lu, Jiating Zhao, Liyuan Liang, Eddy Y. Zeng, and Baohua Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b02586 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Environmental Science & Technology

ACS Paragon Plus Environment


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A stepwise reduction approach reveals mercury competitive binding

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and exchange reactions within natural organic matter

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and mixed organic ligands

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Xujun Liang†,‡,¶, Xia Lu§,‡,¶, Jiating Zhao‡, Liyuan Liang‡, Eddy Y. Zeng†,

6

Baohua Gu‡,£,*

7 8

†

9

and Health, Jinan University, Guangzhou 511443, China

School of Environment and Guangdong Key Laboratory of Environmental Pollution

10

‡

11

37831, USA

12

§

School of Environmental Sciences, Lanzhou University, Lanzhou 730000, China

13

£

Department of Biosystems Engineering and Soil Science, University of Tennessee,

14

Knoxville, TN 37996, USA

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN

15 16

¶ These

authors contributed equally.

17 18 19 20 21

1


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ABSTRACT

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The kinetics of mercuric ion (Hg2+) binding with heterogeneous naturally

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dissolved organic matter (DOM) has been hypothesized to result from competitive

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interactions among different organic ligands and functional groups of DOM for Hg2+.

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However, experimental protocol is lacking to determine Hg2+ binding with various

27

competitive ligands and DOM, their binding strengths, and their dynamic exchange

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reactions. In this study, a stepwise reduction approach using ascorbic acid (AA) and

29

stannous tin [Sn(II)] was devised to differentiate Hg(II) species in the presence of two

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major functional groups in DOM: the carboxylate-bound Hg(II) is reducible by both

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AA and Sn(II), whereas the thiolate-bound Hg(II) is reducible only by Sn(II). Using

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this operational approach, the relative binding strength of Hg2+ with selected organic

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ligands was found in order: dimercaptopropanesulfonate (DMPS) > glutathione (GSH)

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> penicillamine (PEN) > cysteine (CYS) > ethylenediaminetetraacetate (EDTA) >

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citrate, acetate, and glycine at the ligand-to-Hg molar ratio < 2. Dynamic, competitive

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ligand exchanges for Hg2+ from weak carboxylate to strong thiolate functional groups

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were observed among these ligands and within DOM, and the reaction depended on the

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relative binding strength and abundance of thiols and carboxylates, as well as reaction

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time. These results provide additional insights into dynamic exchange reactions of Hg2+

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within multicompositional DOM in controlling the transformation and bioavailability

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of Hg2+ in natural aquatic environments.

42 43

2



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INTRODUCTION

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Natural organic matter consists of thousands of mixed heterogeneous organic

46

compounds,1-3 and is known to play an important role in controlling the chemical

47

speciation and biogeochemical cycling and toxicity of mercury (Hg) in natural aquatic

48

ecosystems.4-11 This effect is in part due to the formation of strong complexes between

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mercuric ion (Hg2+) and a suite of organic ligands, particularly the thiol functional

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groups present in dissolved organic matter (DOM).6,8,12-15 Many low-molecular-weight

51

(LMW) organic ligands have been identified in DOM, such as acetate, citrate, cysteine,

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glutathione, phenol, and glutamate,16-19 although their stability constants (log β) for

53

Hg2+ binding vary by many orders of magnitude.8,14,20,21 In general, Hg2+ forms weaker

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complexes with carboxylate (log β ranging from 3.7 to 23.2) and much stronger

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complexes with thiolate ligands with log β ranging from 26 to 53.1 (Supporting

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Information (SI), Table S1). However, large discrepancies have been reported for the

57

measured log β values for Hg(II)-DOM species (ranging from 10 to 40),4,8,15,22-28 and

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these differences have often been attributed to different origins and properties of DOM

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(e.g., its sulfur or thiol contents), as well as to methodologies and environmental

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conditions used in measurements. Large discrepancies also exist in the reported log β

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values for thiolate binding with Hg2+, such as Hg(II)-cysteine (log β =14.2–43.4) and

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Hg(II)-glutathione (log β =26–41.6) (Table S1).14,20,21,29 However, a recent study using

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Hg LIII-edge EXAFS spectroscopy indicates that the thermodynamic stabilities of

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Hg(NOM-RS)2, Hg(Cys)(NOM-RS) and Hg(Cys)2 in a mixed Hg2+-NOM-Cys system

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are very similar at pH < 7.15 These discrepancies in reported log β values of DOM and

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organic ligands make it difficult to assess their relative binding strengths for Hg2+, how

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they may competitively interact with Hg2+, and what are the dominant Hg(II) species 3


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in order to assess its availability for biological uptake and methylation under realistic

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environmental conditions.

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Unlike bonding between Hg2+ and a single LMW organic ligand,14,15 Hg2+

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complexation with DOM is also known to be kinetically controlled, usually requiring >

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9 h to reach an equilibrium.4, 5, 30 An even longer equilibrium time (> 24 h) was reported

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for Hg(II)-DOM complexation in a contaminated creek water,5,30 as indicated by

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decreasing stannous [Sn(II)]-reducible Hg(II) over time. Similarly, Hg(II) species were

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found to be more bioavailable under non-equilibrium conditions than when Hg2+ and

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DOM had reached pseudo-equilibrium (24 h) prior to exposure to bacteria in a Hg(II)

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uptake study.9 It is therefore hypothesized that newly deposited Hg2+ binds rapidly to

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biologically labile ligands present in the DOM, but over time Hg(II) species becomes

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much less bioavailable, due to a series of ligand exchange reactions between Hg2+ and

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different hypothetical DOM pools within the DOM.4,9 Likewise, competitive ligand

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exchanges for Hg2+ among many different functional groups in DOM, such as weak

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carboxylates and strong thiolates, are thought to be the main cause of the kinetic effect,5,

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30

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species. For example, the Sn(II) reduction technique used in previous studies cannot

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differentiate Hg2+ bound to carboxylate from thiolate. No direct experimental evidence

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has been presented to support the kinetic exchange behavior of Hg(II)-DOM species,

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partly due to technical difficulties in separating or detecting unknown Hg(II)-DOM

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species, particularly at nanomolar Hg(II) concentrations.2, 14

although so far few techniques have been developed to differentiate these Hg(II)

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This study was therefore aimed at devising a technique to differentiate Hg(II)

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species that are bound to carboxylates and thiolates, two typical weak and strong

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binding groups/domains in DOM. This is based on Hg(II) reducibility by a weaker 4



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reducing agent, ascorbic acid (AA), and a stronger reductant, Sn(II) chloride. Using this

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operationally defined, stepwise AA- and Sn(II)-reducible Hg(II) approach as proxies

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for carboxylate- and thiolate-bound Hg(II), we compared relative binding strengths of

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Hg2+ with a suite of LMW organic ligands and their competitive exchange reactions

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among themselves and with DOM. We also investigated the dynamic, competitive

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ligand exchange reactions for Hg2+ from the weak carboxylate to the strong thiolate

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ligands, and then to the strongest non-reducible fractions within DOM over time.

99 100

MATERIALS AND METHODS

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Materials and sample preparation. L-cysteine (CYS), L-penicillamine (PEN),

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glutathione (GSH), glycine (GLY), and 2,3-dimercaptopropanesulfonate monohydrate

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(DMPS) were purchased from Sigma-Aldrich. Disodium ethylenediaminetetraacetate

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(EDTA), citric acid (CTA), and sodium acetate (ACE) were obtained from Fisher

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Scientific. These organic compounds were selected to represent a suite of thiolate and

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carboxylate organic ligands commonly used in Hg(II) biogeochemical investigations.8,

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14, 21, 31, 32

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Hg2+ are summarized in Table S1. Ascorbic acid and SnCl2 were purchased from Fisher

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Scientific and used as reductants for Hg(II). All chemicals and organic ligands are

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certified analytical reagents and were used without further purification.

Their molecular structures, molecular weights, and stability constants with

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Two DOM isolates from terrestrial and aquatic origins were used in this study.

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Elliott soil humic acid (SHA) was obtained from the International Humic Substances

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Society, and EFPC-DOM was isolated from the East Fork Poplar Creek (EFPC) water

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in Oak Ridge, Tennessee, as previously described.2,11 The basic chemical

115

characteristics of these DOM isolates are given in Table S2. All DOM stock solutions 5


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(containing ~ 24 mM C) were prepared in a deoxygenated phosphate-buffered saline

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(PBS) solution at pH 7.4, as used in biological and biochemical investigations.11,33-35

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The PBS consisted of 0.14 M NaCl, 3 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4,

119

as previously described.11, 33-35 The DOM stock solution was filtered through a 0.2-µm

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filter (Acrodisc® with Supor® membrane) before use. Dissolved organic carbon (DOC)

121

concentrations were determined using a Shimazu TOC-5000A total organic carbon

122

analyzer.28

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Stepwise reduction of Hg(II)-ligand complexes. Complexes between Hg2+ and

124

selected organic ligands (ACE, CTA, EDTA, GLY, CYS, PEN, GSH, DMPS) were

125

prepared in 4-mL glass vials by mixing solutions of HgCl2 and organic ligands at a

126

fixed Hg(II) concentration (25 nM), but varying ligand concentrations from 0 to 25 M

127

to yield ligand-to-Hg molar ratios of 0–1000 in PBS. All sample preparations were

128

performed in an anoxic chamber under N2 (~98%) and H2 (~2%) (Coy Lab Products,

129

Grass Lake, MI), and all vials were sealed with PTFE-lined silicone screw caps and

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then placed on a rotary shaker (90 rpm). Samples were equilibrated for about 1 h, which

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was sufficient to reach an equilibrium based on our preliminary results and previous

132

studies,14,15 and were then removed from the anoxic chamber for analysis. An aliquot

133

(100 µL) was immediately transferred to a clean 4-mL vial prefilled with 800 µL

134

deionized water. This was followed by adding an excess amount of AA (100 µL of 1

135

mM AA stock) to reduce Hg(II), and subsequent detection of the purgeable gaseous

136

Hg(0) directly using a Hg(0) analyzer (Lumex RA-915+, Ohio Lumex Co.).6,33,36,37

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After completion of the AA reduction (or when Hg(0) was non-detectable), 100 µL

138

SnCl2 (20% SnCl2 in 10% HCl) was immediately added to the remaining sample and

139

again analyzed for Hg(0) by Lumex RA-915+. Herein, the AA-reducible Hg(II) and 6



140

Sn(II)-reducible Hg(II) are respectively denoted as HgR-AA and HgR-Sn throughout

141

the text. Separately, another aliquot (100 µL) of the sample was transferred to a 4 mL

142

clean vial prefilled with 850 µL deionized water and 50 µL BrCl (5%, v/v), and samples

143

were equilibrated overnight at 4°C and analyzed for total Hg(II) (HgT).5,33,38

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Similar experiments were performed for DOM bound Hg(II) species, except

145

that samples were pre-equilibrated for either 1 or 24 h before measurement of HgR-AA

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and HgR-Sn, since Hg(II)-DOM complexation is known to be time dependent.4, 5, 30

147

While the Hg(II) concentration was kept constant (25 nM), DOM concentrations varied

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from 0 to 1 mM C (equivalent to DOC:Hg molar ratios of 0 to 40,000).23-25 Additionally,

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since DOM is known to reduce a small amount of Hg(II),6, 38 all samples were purged

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with ultrapure N2 to remove purgeable gaseous Hg(0), HgP, before measuring HgR-

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AA and HgR-Sn. For clarity of presentation and comparisons, these data were

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normalized to the total non-purgeable Hg(II) (HgNP) remaining in the system since

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HgP varied slightly with different concentrations of DOM. All samples were prepared

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in duplicate in batch experiments and, in some cases, batch experiments were repeated

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2–3 times for quality assurance. A good mass balance was obtained during sample

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preparation and complexation experiments (Figures S1 and S2). Average

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concentrations are reported, and error bars in figures represent one standard deviation

158

of all replicate samples. In the case of duplicate measurements, error bars represent the

159

deviation between the two replicates (X1 and X2), defined as the absolute value of (X1–

160

X2)/2.39

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Competitive ligand exchange reactions with Hg2+. Ligand exchange reactions were

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subsequently performed to evaluate relative binding strengths of selected organic

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ligands and their competitive interactions with Hg2+. To study if carboxylate can 7


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exchange thiolate ligands for Hg2+, solutions of Hg2+-thiolate complexes were first

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prepared with molar ratios of DMPS:Hg and GSH:Hg at 1:1 and CYS:Hg at 2:1 in PBS,

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and equilibrated for about 1 h in a series of 4-mL glass vials under anoxic conditions.

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Different thiol:Hg ratios were used to ensure that at the selected minimal thiol

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concentrations, the complexed Hg(II) was mostly non-reducible by AA. Therefore,

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when weaker-binding ligands such as CTA and EDTA were added, the exchanged

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Hg(II), if any, was expected to be reducible or detectable by AA reduction. CTA and

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EDTA were added at varying concentrations (up to a ligand:Hg ratio of 1000), and the

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amounts of Hg(II) exchanged by CTA or EDTA were determined after either 1 or 24 h

173

equilibration, using the stepwise reduction technique. Similarly, CYS and GSH at

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varying concentrations (up to a ligand:Hg ratio of 4) were added to the pre-prepared

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DMPS:Hg solutions to study if CYS and GSH could exchange DMPS for Hg2+. Here

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DMPS is the strongest ligand among all the organic ligands studied. The amounts of

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Hg(II) exchanged by CYS or GSH were determined after 1 or 24 h equilibration to

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observe whether the exchange reactions were time-dependent.

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Exchange reactions between LMW ligands and the two DOM isolates were also

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conducted to improve our understanding of competitive interactions within DOM over

181

time. In this case, Hg(II)-DOM complexes were first prepared in solutions with a

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DOC:Hg molar ratio of ~19,000, typical of the natural aquatic environment.10, 40 The

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solution was equilibrated for either 1 or 24 h in an anoxic chamber. Before the addition

184

of competing organic ligands, DOM reducible Hg(II) was determined by purging with

185

ultrapure N2 to remove gaseous Hg(0) (Figure S3), as noted above. After purging,

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samples were placed back in the anoxic chamber and spiked with varying

187

concentrations of competing ligands, including CYS, GSH, DMPS, CTA and EDTA, 8



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at varying ligand:Hg ratios, based on their relative binding strengths. After an additional

189

1 h equilibration, samples were taken out of the chamber and analyzed for the

190

exchanged Hg(II) by the stepwise reduction technique. All results were again

191

normalized to the total HgNP (i.e., Hg(II) remaining in solution).

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RESULTS AND DISCUSSION

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Differentiating Hg(II)-carboxylate and thiolate complexes by stepwise reduction

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To determine interactions between Hg2+ and various organic ligands, we first developed

196

a reduction scheme to differentiate Hg(II)-carboxylate (–COO-) and thiolate (–S-)

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complexes, using a suite of natural and synthetic organic ligands commonly used in

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Hg(II) biogeochemical investigations.8,14,21,31,32 Carboxylate/amine and thiolate

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compounds were selected to represent the most abundant and important functional

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groups of DOM for binding with Hg2+ in the natural environment.5,8,10,28 Results

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indicate that at ligand:Hg molar ratios < 5, more than 90% of the Hg(II)-carboxylate

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species (ACE, CTA, EDTA and GLY) were reduced by AA (Figure 1a), whereas

203

negligible amounts of Hg(II) ( 2 (Figure 1a), indicating that Hg2+ forms

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stronger bonds with thiolate than with carboxylate ligands, as expected.5,8,10,28 However,

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all Hg(II)-thiolate species, except Hg(II)-DMPS, were completely reducible when Sn(II)

208

was used as a reductant (Figure 1b). For Hg(II)-DMPS, ~75% of the Hg(II) was non-

209

reducible at the DMPS:Hg ratio of 1, but this fraction decreased to ~60% at DMPS:Hg

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ratios ≥3. This result is explained by the formation of binuclear bidentate Hg2(DMPS)2

211

species at the DMPS:Hg molar ratio of 1, but formation of mixed Hg2(DMPS)2 and 9


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mono-nuclear tetra-dentate, Hg(DMPS)4 complexes at higher DMPS:Hg ratios.41 The

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Hg2(DMPS)2 complex forms a ring-structure and is among the most stable since its Hg-

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S bond distance is shorter than those of Hg(DMPS)4 species.41 Regardless of its

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structure, DMPS formed the strongest Hg(II)-thiolate species, as they were the most

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resistant to reduction by both Sn(II) and AA. Figure 1a also shows that, at the thiol:Hg

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ratio of 1, < 5% of the Hg(II) species in the presence of GSH were reducible by AA,

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but ~35% and 50% of the Hg(II) species in the presence of PEN and CYS were AA-

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reducible. The result indicates that the amounts of Hg2+ bound to thiol ligands that are

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non-AA-reducible are in order: GSH > PEN > CYS.

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While all Hg(II)-carboxylate complexes are reducible by AA at the ligand:Hg

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ratio < 5 (Figure 1a), increasing EDTA:Hg ratios decreased HgR-AA (Figure 1c). At

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the EDTA:Hg ratio of 1000, HgR-AA was essentially non-detectable. However, ACE,

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GLY or CTA bound Hg2+ was completely reducible by AA, even at the ligand:Hg ratio

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of 1000 (Figure 1c), indicating that Hg2+ forms stronger complexes with EDTA than

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with ACE, CTA, and GLY. This finding is supported by the formation of multidentate

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Hg(II)-EDTA complexes via both carboxyl and amine functional groups, since EDTA

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is a known metal chelator.42 However, when Sn(II) was used as a reducing agent, all

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carboxylate-bound Hg2+, including Hg-EDTA, were completely reducible, irrespective

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of the ligand:Hg ratio (up to 1000) (Figure 1b, d). Together, based on the amounts of

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AA- or Sn(II)-reducible Hg(II) bound to each ligand at the thiol:Hg ratio PEN > CYS > EDTA >

234

CTA, ACE, GLY. These results suggest that the stepwise AA and Sn(II) reduction

235

approach could offer a means to qualitatively discern relative binding strengths of 10



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ligands for Hg2+, particularly the two major functional groups of carboxyls and thiols

237

observed in natural organic matter.

238

Competitive ligand-exchange reactions

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To demonstrate the application of the stepwise AA and Sn(II) reduction approach, we

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subsequently investigated Hg(II) competitive ligand exchange reactions between

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selected carboxylate (CTA and EDTA) and thiolate (CYS and GSH) ligands, and

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between these and DMPS, the strongest Hg(II)-binding ligand observed in this study.

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Results show that, when CTA was added to CYS:Hg (2:1) or GSH:Hg (1:1) pre-

244

equilibrated solutions, HgR-AA remained low or unchanged (Figure 2a). No ligand

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exchange occurred even with the addition of 500–1000 times more CTA than CYS or

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GSH, indicating that Hg(II)-thiol complexes are strong and the thiolates cannot be

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exchanged by CTA, as expected. Similarly, at the EDTA:Hg ratio of 3, EDTA could

248

not replace CYS or GSH. However, at EDTA:Hg ratios of 500 and 1000, HgR-AA

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decreased to near zero (Figure 2a), whereas HgR-Sn remained unchanged (Figure 2b).

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In these cases, neither AA nor Sn(II) reduction could differentiate whether the Hg(II)

251

is complexed with thiols or EDTA since all Hg(II) complexes with CYS, GSH or EDTA

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(at high EDTA:Hg ratios) were non-reducible by AA (Figure 1a,c) but completely

253

reducible by Sn(II) (Figure 1b,d).

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We then examined ligand exchange reactions between the strongest Hg(II)-

255

binding ligand DMPS and CTA, EDTA, CYS, or GSH at varying ligand:Hg ratios.

256

Again, no ligand exchange was observed with CTA (up to a CTA:Hg ratio of 1000) or

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with EDTA (at the EDTA:Hg ratio of < 3) after 1 h or 24 h reactions (Figure 2c). With

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the addition of CYS or GSH (at ligand:Hg ratios of 1–4) or excess amounts of EDTA

259

(at EDTA:Hg ratios of 20–1000), HgR-AA remained low to non-detect since AA 11


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cannot reduce these Hg(II) species (Figure 2), as noted above. But using Sn(II)

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reduction, a substantially increased HgR-Sn was observed following the addition of

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CYS, GSH, or EDTA (at EDTA:Hg ratios > 20; Figure 2d), indicating that Hg(II) bound

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to DMPS was exchanged by CYS, GSH, or EDTA. With the addition of only 50 nM

264

CYS (or CYS:Hg ratio = 2), HgR-Sn increased from ~23% to 55%, and to 90% at the

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CYS:Hg ratio of 4. Similarly, HgR-Sn increased to about 63% and 87% with the

266

addition of 25 and 100 nM GSH (or GSH:Hg ratios of 1 and 4), respectively. EDTA

267

was also effective in exchanging DMPS at relatively high EDTA:Hg ratios (Figure 2d).

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HgR-Sn remained at ~23% at the EDTA:Hg ratio of 3, but increased to 40%, 55%, and

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82% when EDTA:Hg ratios increased to 20, 200, and 1000, respectively. These

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findings indicate that, despite DMPS forms strong complexes with Hg2+(Figure 1b),

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CYS, GSH, and EDTA at relatively high concentrations can competitively displace

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DMPS, likely by breaking the ring structure of Hg2(DMPS)2 formed at the DMPS:Hg

273

molar ratio of 1.41 The resulting product is a mixed DMPS-Hg(II)-ligand species (i.e.,

274

CYS, GSH and EDTA), which is less stable than Hg2(DMPS)2 and thus readily reduced

275

by Sn(II). However, the amounts of Hg(II) exchanged by EDTA were unexpectedly

276

higher than those predicted by the thermodynamic speciation model (Table S3). The

277

model predicts that Hg(II) is completely associated with DMPS even at the EDTA:Hg

278

ratio of 1000, because the reported log β values for Hg(II)-DMPS species are about 19

279

orders of magnitude higher than that of Hg(II)-EDTA (Table S1). Similarly, model

280

shows that Hg(II) is predominantly bound by DMPS in the presence of CYS or GSH

281

both at 4 times of the DMPS concentration (Table S3), which differs from our

282

experimental observations. These results therefore question the validity of some of the

283

reported log β values. Additional work is needed to improve log β measurements for 12



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Hg2+ binding with various organic ligands (Table S1), as noted earlier.14,20,21,29 For

285

example, the reported log β value for Hg(II)-CYS was similar or even greater than

286

Hg(II)-GSH (Table S1), but our data (Figures 1 and 2) indicate that more Hg2+ is

287

associated with GSH (that is non-AA-reducible) than with CYS at the ligand:Hg ratio

288

< 2. These results again illustrate the novel application of the stepwise reduction

289

approach for comparing relative binding strengths of organic ligands and determining

290

their competitive exchange reactions with Hg2+.

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We observed no significant differences in exchange reactions when the reaction

292

time increased from 1 to 24 h (Figure 2c,d), indicating that these ligand exchange

293

reactions are rapid, and generally complete < 1 h. This result is consistent with previous

294

13C

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between free thiols and the thiolate bound to Hg(II) are usually within seconds.43, 44

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These reactions are also reversible, depending on the binding strength and relative

297

concentrations of the ligand in solution. Similarly, a recent study of Hg(II) ligand

298

exchange between CYS and thiol groups in Suwanee River DOM showed that chemical

299

equilibrium between Hg-CYS and Hg-DOM species could be achieved within minutes

300

of reaction.15

301

Hg(II)-DOM complexation dynamics

302

Using the same approach, we also investigated the dynamics of Hg2+ binding with two

303

major functional groups (i.e., –COO- and –S-) in two DOM isolates: The SHA was used

304

to represent a high molecular weight, high aromaticity terrestrially derived DOM,

305

whereas EFPC-DOM was used to represent aquatic DOM with a relatively low

306

molecular weight and aromaticity.2,

307

semiquinone moieties acting as weak reducing agents, a small amount of Hg(II) was

and 1H NMR spectroscopic studies, which showed that Hg(II) exchange reactions

6, 38

Since all DOM contains some levels of

13


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reduced to elemental Hg(0) by DOM itself upon mixing (Figures S2), as previously

309

reported.6, 30, 38 Slightly more Hg(II) was reduced by SHA than EFPC-DOM, especially

310

after 24 h reaction, since EFPC-DOM contains a higher S or thiol content (Table S2)

311

and thus forms more Hg(II)-thiol complexes which could not be reduced by the

312

semiquinone compounds in DOM.6,13,15,31,45 Therefore, lower Hg(II) reduction was

313

observed with increasing EFPC-DOM concentrations or DOM:Hg ratios and with

314

increasing reaction time, as more Hg(II) became complexed with thiol functional

315

groups in EFPC-DOM than SHA (Figure S2).

316

Following the removal of Hg(0) by purging, HgR-AA, HgR-Sn, and non-

317

reducible Hg(II) (HgNR) were analyzed in sequence, and the dynamics of Hg(II)

318

complexation and competitive ligand exchange among different DOM functional

319

groups were determined with time and varying DOM:Hg ratios (Figure 3). Since the

320

amount of purgeable Hg(0) varied slightly with the addition of different amounts of

321

DOM (Figure S2), data in Figure 3 were normalized to total non-purgeable Hg(II)

322

(HgNP) in the system for comparison. Results indicate that HgR-AA decreased

323

continually with increasing DOM concentrations, and lower amounts of HgR-AA were

324

produced in the presence of EFPC-DOM than SHA due to a higher thiol content of

325

EFPC-DOM (Table S2). Additionally, sharp decrease in HgR-AA was observed when

326

Hg(II) was pre-equilibrated with DOM for a longer time (24 h). For example, with 1-h

327

pre-equilibration, HgR-AA in EFPC-DOM (at 0.1 mM C) was ~27%, but decreased to

328

zero when EFPC-DOM-Hg pre-equilibrated for 24-h (Figure 3a,b). For SHA under

329

same conditions, HgR-AA (at 0.1 mM C) decreased from ~65% to 45%, and further

330

decreased to zero at the DOM concentration above 0.5 mM C (Figure 3c,d). Coincident

331

with decreasing HgR-AA, HgR-Sn increased with DOM concentration, reached a 14



332

maximum and then decreased with increasing DOM concentration or DOM:Hg ratio.

333

This is especially evident for samples that were pre-equilibrated for 24 h, although the

334

HgR-Sn maxima varied with DOM isolates and the reaction time during pre-

335

equilibration (Figure 3b,d). In the same experiments, a consistently increased non-

336

reducible HgNR was observed with increasing DOM concentrations, as HgNR is the

337

most stable Hg(II)-DOM species that can neither be reduced by Sn(II) nor AA.

338

The dynamic changes in HgR-AA, HgR-Sn, and HgNR with time and DOM

339

concentrations (Figure 3) indicate competitive binding and exchange reactions between

340

different pools or functional groups of DOM for Hg2+, as previously hypothesized,5, 6,

341

46

342

concentration (e.g., < 0.2 mM C), most Hg(II) may be bound to carboxylate initially in

343

SHA (at 1 h), and could therefore be reduced by AA (Figure 3c). However, with

344

increasing reaction time (24 h; Figure 3d), HgR-AA decreased, whereas HgR-Sn and

345

HgNR increased, suggesting that some weakly-bound Hg(II)-carboxylate species were

346

exchanged with thiol ligands, or formed strong Hg(II)-thiolate complexes or other non-

347

reducible Hg(II) species. In the case of EFPC-DOM, a shorter reaction time (pre-

348

equilibrated for 1 h) resulted in the production of ~ 25% HgR-AA, but most Hg(II)

349

(~75%) was bound to thiolates when the DOM concentration is at or greater than 0.2

350

mM C (Figure 3a). However, no HgR-AA could be detected for samples pre-

351

equilibrated for 24 h even at a low DOM concentration (0.1 mM C) (Figure 3b). The

352

result suggests that all weakly-bound Hg(II) had been replaced by strongly-bound

353

thiolate or non-reducible Hg(II) species within 24 h. However, HgR-Sn increased and

354

reached a maximum of ~65% (at 0.1 mM C). It then decreased, while HgNR increased

355

with increasing DOM concentrations. These observations again indicate time-

but have not been experimentally validated. Our results suggest that, at a low DOM

15


Page 16 of 32

Page 17 of 32


356

dependent Hg(II) exchange from the carboxyl to the thiol functional groups in DOM,

357

and then to the non-reducible pool of DOM. Similar trends were observed with SHA

358

(Figure 3c,d), although the HgR-Sn maximum occurred at a higher DOM concentration

359

(~ 0.35 mM C), which can be attributed to a lower S or thiol content in SHA than EFPC-

360

DOM, as discussed above (Table S2). Therefore, the HgR-Sn maxima depend not only

361

on the reaction time and the DOM:Hg ratio, but also on the type of DOM and its thiol

362

content.

363

Competitive ligand exchange reactions with DOM

364

Although DOM forms exceptionally strong complexes with Hg2+,6,10,13,15 it remains

365

unclear to what extent the DOM-bound Hg(II) may be exchanged by competing organic

366

ligands, such as CTA, EDTA, CYS, GSH, and DMPS. We therefore further

367

investigated the dynamics of competitive exchange reactions between various

368

carboxylate or thiolate ligands and DOM that had been pre-equilibrated with Hg(II) for

369

either 1 h or 24 h. Based on their relative binding strengths (Figure 1), a low thiol:Hg

370

ratio (either 1 or 2) but a high CTA:Hg or EDTA:Hg ratio (1000) was used in these

371

studies. The measured HgR-AA, HgR-Sn, and HgNR (Figure 4) were also normalized

372

to the total HgNP after removing Hg(0) following its pre-equilibration with DOM

373

(Figure S3), as described above. No HgR-AA was observed for Hg2+ complexed with

374

EFPC-DOM 1 h after the addition of EDTA, CTA, CYS, GSH, and DMPS (Figure 4a).

375

However, about 25% HgR-AA was observed for Hg(II) complexed with SHA (pre-

376

equilibrated for 1 h) either with or without addition of CTA (Figure 4b), again

377

indicating that CTA is ineffective in competitively binding or exchanging Hg2+ that had

378

been bound with DOM. A slightly lower amount of HgR-AA (~9%) was observed for

16



379

Hg(II) complexed with SHA after 24 h pre-equilibration, as more Hg2+ became

380

complexed with thiol functional groups or shifted to the HgNR pool over time.

381

However, HgR-Sn increased 10-15% with the addition of CYS, GSH, and

382

EDTA but decreased (up to 25%) with the addition of DMPS when Hg2+ was pre-

383

equilibrated with DOM for 1 h (Figure 4c,d). The increase in HgR-Sn suggests that a

384

fraction of HgNR was exchanged by CYS, GSH, or EDTA and thus reduced by Sn(II).

385

Both CYS and GSH appeared equally effective in exchanging DOM functional groups

386

for Hg2+ although a lower amount of GSH (GSH:Hg = 1:1) was used than CYS

387

(CYS:Hg = 2:1). A slightly lower amount of HgR-Sn was observed with the addition

388

of EDTA at a high EDTA:Hg ratio of 1000, in line with the binding strength of these

389

ligands established above in the order GSH > CYS > EDTA (Figure 1). However, the

390

addition of DMPS substantially decreased HgR-Sn but increased HgNR, especially for

391

SHA (Figure 4c-f), indicating DMPS replacement of Hg2+ that had been bound to DOM

392

thiolates. The result implies that the HgNR pool could, at least partially, be attributed

393

to the formation of stable chelated Hg(II) species such as Hg(II)-DMPS. Such exchange

394

reactions appeared lower (< 15%) on EFPC-DOM than SHA, particularly after samples

395

were pre-equilibrated for a longer time (Figure 4c,d). When Hg(II) was pre-equilibrated

396

with EFPC-DOM for 24 h, no differences in HgR-Sn were observed (Figure 4c),

397

regardless of whether or not there was addition of CTA, EDTA, CYS, GSH or DMPS.

398

It suggests that all Hg(II) had been complexed with strong binding sites in EFPC-DOM,

399

and no Hg2+ could be exchanged or replaced by the competing ligands. These results

400

are indicative of the transfer of Hg2+ from weak to strong, and to stronger, nonreducible

401

binding sites within DOM macromolecules, making Hg2+ less or non-exchangeable

402

over time. 17


Page 18 of 32

Page 19 of 32


403

ENVIRONMENTAL IMPLICATIONS

404

The present study reveals that stepwise AA and Sn(II) reduction could offer a means to

405

assess relative binding strengths of Hg2+ with a suite of organic ligands, such as DMPS

406

> GSH > PEN > CYS, based on the amounts of Hg(II) bound to each ligand that are

407

non-AA-reducible at the ligand:Hg molar ratio < 2. This order of Hg2+ binding strengths

408

with thiol ligands differs from that calculated based on reported log values, which vary

409

by many orders of magnitude,20,21,47-49 and also deviates from recently determined

410

stability constants for HgL2 species with the following ligands (L): GSH (log  = 38.8),

411

PEN (log  = 36.9), and CYS (log  = 37.5).14 However, the order is consistent with

412

the effects of these thiols on Hg(II) methylation by certain anaerobic bacteria, such as

413

G. sulfurreducens PCA and D. desulfuricans ND132, assuming that bacteria take up

414

Hg(II) via ligand exchange with thiols in solution.11, 50 While many biochemical factors

415

may influence the rate and extent of Hg(II) methylation, our results offer a reasonable

416

explanation as bacterial cells have to compete with thiols for Hg2+ binding and uptake

417

in solution.50, 51 Among the thiol ligands examined, CYS forms the weakest Hg(II)

418

complexes and shows to enhance Hg(II) methylation by G. sulfurreducens PCA,32, 34,

419

52, 53

420

are shown to enhance Hg(II) methylation by D. desulfuricans ND132, since ND132

421

cells carry more thiol functional groups and are more competitive in Hg(II) sorption

422

and uptake than PCA cells.32, 36 Similarly, since EFPC-DOM binds strongly with Hg2+,

423

it inhibits Hg(II) uptake and methylation by G. sulfurreducens PCA cells but not by D.

424

desulfuricans ND132.11 DMPS forms the strongest Hg(II) complexes and thus inhibits

425

Hg(II) uptake and methylation by both PCA and ND132 cells.11, 36

whereas all other thiols inhibit its methylation. However, all thiols except DMPS

18



426

Importantly, EDTA at relatively high concentrations (or high EDTA:Hg ratios)

427

is capable of competing with thiols, such as DMPS, for Hg2+ binding (Figure 2d),

428

despite that the reported stability constant of Hg(II)-DMPS is 19 orders of magnitude

429

higher than that of Hg(II)-EDTA (Table S1).7,13,19 Speciation calculations indicate that

430

EDTA, even at the concentration of 1000× greater than DMPS, is unable to replace

431

DMPS for Hg(II) binding (Table S3). These results suggest that Hg2+ interaction with

432

DOM may be more complicated than previously thought, as DOM contains abundant

433

carboxyl and amine functional groups (in addition to thiols). DOM compounds

434

containing both carboxylates and amines with certain configurations (like EDTA) at

435

relatively high concentrations could form strong complexes with Hg2+, although

436

experimental studies and speciation calculations so far have shown that thiolates are the

437

dominant functional groups in DOM responsible for Hg2+ binding.6,10,13,15,24 Similarly,

438

GSH can form stronger complexes with Hg(II) than CYS and PEN (Figure 1a),

439

resulting from multi-coordination involving cysteinyl sulfur and amine in GSH at a low

440

thiol:Hg ratio (< 2).29 Although additional investigations are required to determine

441

whether such a relationship holds at higher thiol:Hg ratios (> 2), these exchange

442

reactions could occur in natural water and DOM where many individual thiol ligands

443

likely exist at very low concentrations (or at the thiol:Hg ratio < 2), as DOM consists

444

of thousands of individual molecules.1, 2, 3 Therefore, the chemical structure and relative

445

abundance of thiol, carboxyl, and amine functional groups in DOM may ultimately

446

dictate Hg(II)-ligand binding and its chemical speciation in natural aquatic

447

environments.

448

Time-dependent competitive ligand exchange reactions for Hg2+ in DOM were

449

illustrated by dynamic changes in HgR-AA, HgR-Sn, and HgNR pools within 19


Page 20 of 32

Page 21 of 32


450

heterogeneous DOM macromolecules (Figure 3). At the DOC:Hg molar ratios of 4,000

451

to 40,000,10, 24 HgR-AA was found to generally decrease whereas HgNR increase with

452

time, as Hg2+ is likely exchanged from carboxyl to thiol and finally to stronger binding

453

functional groups/domains, making Hg(II) non-reducible by Sn(II). The shorter the

454

reaction time between Hg2+ and DOM, the more Hg2+ may be complexed with

455

carboxylates and thus more readily reduced by AA or Sn(II). However, increasing

456

reaction time results in increase in the HgNR pool in DOM, supporting the hypothesis

457

that the reducibility of Hg(II) bound to DOM decreases with time as a result of Hg(II)

458

rearrangement or exchange reactions from abundant weak binding sites (e.g., carboxyls

459

and amines) to strong thiolate binding sites in DOM.5, 30, 46, 54 These observations are

460

consistent with the observed reduction rate of Hg(II)-thiolates nearly 2 orders of

461

magnitude slower than that of Hg(II)-carboxylate complexes.54 The results also agree

462

with findings that Hg(II) is more bioavailable for microbial uptake under

463

nonequilibrium conditions (a short reaction time) than when Hg2+ and DOM have

464

reached pseudoequilibrium (24 h) prior to cell exposure.9

465

The stepwise reduction approach enables experimental determination of

466

competitive ligand-exchange reactions, and it shows that the complexed Hg(II) is

467

exchangeable among LMW organic ligands and between DOM and LMW ligands.

468

Increase in thiol concentrations such as CYS and GSH could release Hg(II) that had

469

been bound to DMPS or DOM although their binding strengths with Hg2+ may be

470

higher than those of CYS and GSH. These results support that Hg(II) chemical

471

speciation in natural aquatic systems may change constantly with input of new organic

472

ligands and could thus make Hg(II) species more or less available for biological uptake

473

and methylation. For example, enhanced Hg(II) methylation has been observed in 20



474

benthic biofilms and green alga periphyton, likely due to increased levels of LMW thiol

475

ligands.19, 55 Similarly, a positive correlation is found between LMW thiols and Hg(II)

476

in lake periphytic biofilms.56 In line with these studies, high resolution mass

477

spectrometry examination reported compositional changes in organic ligands and in

478

Hg(II) speciation during freshwater algal growth under different lighting conditions.19

479

Phytoplankton-derived organic ligands are also shown to significantly enhance Hg(II)

480

methylation during planktonic blooms, although the authors attributed this effect to an

481

overall increased bacterial activity.18 Our results, as well as others,19, 55, 57 suggest that

482

changes in Hg(II) speciation due to input of new organic ligands during algal blooms

483

could be partially responsible. Molecular compositional changes of DOM and its

484

dynamic equilibrium with Hg(II) could thus be critically important in controlling the

485

transformation and bioavailability of Hg(II) in natural aquatic ecosystems.

486 487

ASSOCIATED CONTENT

488

Supporting Information

489

The Supporting Information is available free of charge on the ACS Publications website

490

at DOI: XXXX.

491

Molecular structure of organic ligands (L) used in this study and their binding

492

stability constants (log β) with Hg2+ (Table S1); Elemental composition and

493

characteristics of dissolved organic matter (DOM) isolates, soil humic acid (SHA)

494

and EFPC-DOM, used in this study (Table S2); Speciation calculations of Hg(II) (25

495

nM) reactions with DMPS (25 nM) with or without added competing ligands

496

(EDTA, CYS, and GSH) (Table S3); Total Hg (HgT) determination during stepwise

497

reduction of Hg(II)-ligand complexes (Figure S1); Analyses of purgeable Hg(II) 21


Page 22 of 32

Page 23 of 32


498

(HgP), nonpurgeable Hg(II) (HgNP), and total Hg (HgT) (Figure S2); Mass balance

499

analyses of purgeable Hg (HgP) and nonpurgeable Hg(II) (HgNP) (Figure S3);

500

Determination of Hg(II) reduction or purgeable Hg(0) (HgP) in PBS by citric acid

501

(CTA) (Figure S4).

502

AUTHOR INFORMATION

503

Corresponding Author

504

* Telephone: (865)-574-7286; E-mail: [email protected]

505

Notes

506

The authors declare no competing financial interest.

507 508

ACKNOWLEDGMENTS

509

We thank X. Yin, L. Zhao, L. Wang, and X. Yin for technical assistance in laboratory

510

preparations and sample analyses. This research was sponsored in part by the Office of

511

Biological and Environmental Research within the Office of Science of the U.S.

512

Department of Energy (DOE), as part of the Mercury Science Focus Area project at the

513

Oak Ridge National Laboratory (ORNL) and by China Postdoctoral Science program

514

(2017M622906). The Department of Energy will provide public access to these results

515

of federally sponsored research in accordance with the DOE Public Access Plan

516

(http://energy.gov/downloads/doe-public-access-plan). ORNL is managed by UT-

517

Battelle, LLC under Contract No. DE-AC05-00OR22725 with DOE.

518 519 520 521 522

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48. Casas, J. S.; Jones, M. M., Mercury(II) complexes with sulfhydryl containing chelating agents: stability constant inconsistencies and their resolution. J. Inorg. Nucl. Chem. 1980, 42, (1), 99-102. 26



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49. Oram, P. D.; Fang, X.; Fernando, Q.; Letkeman, P.; Letkeman, D., The formation constants of mercury(II)−glutathione complexes. Chem. Res. Toxicol. 1996, 9, (4), 709-712.

678 679 680 681

50. Thomas, S. A.; Tong, T.; Gaillard, J.-F., Hg(II) bacterial biouptake: the role of anthropogenic and biogenic ligands present in solution and spectroscopic evidence of ligand exchange reactions at the cell surface. Metallomics 2014, 6, (12), 2213-2222.

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51. Dunham-Cheatham, S.; Mishra, B.; Myneni, S.; Fein, J. B., The effect of natural organic matter on the adsorption of mercury to bacterial cells. Geochim. Cosmochim. Acta 2015, 150, 1-10.

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52. Schaefer, J. K.; Morel, F. M. M., High methylation rates of mercury bound to cysteine by Geobacter sulfurreducens. Nat. Geosci. 2009, 2, 123.

687 688 689

53. Lin, H.; Lu, X.; Liang, L.; Gu, B., Cysteine inhibits mercury methylation by Geobacter Sulfurreducens PCA mutant ΔomcBESTZ. Environ. Sci. Technol. Lett. 2015, 2, 144–148.

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Page 28 of 32

Page 29 of 32


707

100

80 DMPS GSH CYS PEN ACE CTA EDTA GLY

60 40 20 0

20

1

2

(c)

3

ACE

CTA

(b)

4

5

0

EDTA

GLY

(d)

80

100

HgR-Sn (% of HgT)

HgR-AA (% of HgT)

40

0

60 40 20

1

2

3

4

5

80 60 40 20

0

0 20

708

60

(a) 0

100

80

HgR-Sn (% of HgT)

HgR-AA (% of HgT)

100

50

200

500

1000

20

Ligand:Hg(II) molar ratio

50

200

500

1000

Ligand:Hg(II) molar ratio

709 710 711

Figure 1. Determination of ascorbic acid-reducible Hg(II) (HgR-AA) (a, c) and Sn(II)-

712

reducible Hg(II) (HgR-Sn) (b, d) that are bound to various natural and synthetic organic

713

ligands, including acetate (ACE), citric acid (CTA), ethylenediaminetetraacetate

714

(EDTA), glycine (GLY), L-cysteine (CYS), L-penicillamine (PEN), glutathione (GSH),

715

and 2,3-dimercaptopropanesulfonate (DMPS) at varying ligand:Hg(II) molar ratios in

716

PBS (pH = 7.4).

717 718

28



(a) HgR-AA No EDTA/CTA +CTA/Hg(II)=3 +CTA/Hg(II)=500 +CTA/Hg(II)=1000 +EDTA/Hg(II)=3 +EDTA/Hg(II)=500 +EDTA/Hg(II)=1000

80

60

10

100

HgR-Sn (% of HgT)

HgR-AA (% of HgT)

100

CYS/Hg(II)=2 (c) HgR-AA

DMPS/Hg(II)=1 +CYS/Hg(II)=2 +CYS/Hg(II)=4 +GSH/Hg(II)=1 +GSH/Hg(II)=4 +CTA/Hg(II)=3 +CTA/Hg(II)=500 +CTA/Hg(II)=1000 +EDTA/Hg(II)=3 +EDTA/Hg(II)=20 +EDTA/Hg(II)=200 +EDTA/Hg(II)=1000

80

60

10

719

60

40

100

CYS/Hg(II)=2

GSH/Hg(II)=1

(d) HgR-Sn

80

60

40

20

5 0

80

0

GSH/Hg(II)=1

HgR-Sn (% of HgT)

HgR-AA (% of HgT)

100

(b) HgR-Sn

20

5 0

Page 30 of 32

1

Exchange time (h)

0

24

1

Exchange time (h)

24

720 721 722

Figure 2. Competitive ligand exchange for Hg2+ in (a, b) Hg(II)-thiol complexes (CYS

723

and GSH) by CTA or EDTA, and (c, d) Hg(II)-DMPS by CTA, EDTA, CYS, or GSH

724

at varying ligand concentrations by stepwise measurements of HgR-AA and HgR-Sn.

725

Complexes of Hg-CYS, Hg-GSH, and Hg-DMPS were formed by pre-equilibrating

726

corresponding Hg-Ligand solutions for ~1 h, followed by the addition of competing

727

ligands. Exchange reaction times were set at 1 h for Hg-CYS and Hg-GSH complexes

728

(a, b) and 1 and 24 h for the Hg-DMPS complex (c, d). All reactions were performed

729

in PBS (pH = 7.4). See main text for additional details.

730

29


Page 31 of 32


731 732 733 734

Figure 3. Hg(II) species distributions, normalized to the total non-purgeable Hg(II)

735

(HgNP), during stepwise AA and Sn(II) reduction of Hg(II) that has been pre-

736

equilibrated with varying concentrations of EFPC-DOM (a, b) or SHA (c, d) in PBS

737

(pH = 7.4) for either 1 h or 24 h. HgR-AA represents AA-reducible Hg(II), and HgR-

738

Sn represents Sn(II)-reducible Hg(II). Non-reducible Hg (HgNR) denotes Hg(II) that

739

cannot be reduced by either AA or Sn(II).

30



100 (b) HgR-AA_SHA

HgR-AA (% of HgNP)

100 (a) HgR-AA_EFPC-DOM DOC only +CYS/Hg(II)=2 +GSH/Hg(II)=1 +DMPS/Hg(II)=1 +CTA/Hg(II)=1000 +EDTA/Hg(II)=1000

80 60

80 60 40

4 2

20

0

0 100 (d) HgR-Sn_SHA

HgR-Sn (% of HgNP)

100 (c) HgR-Sn_EFPC-DOM 80

80

60

60

40

40

20

20

0

0 100 (f) HgNR_SHA

HgNR (% of HgNP)

100 (e) HgNR_EFPC-DOM 80

80

60

60

40

40

20

20

0

1

Page 32 of 32

0

24

EFPC_DOM-Hg(II) binding time (h)

1

SHA-Hg(II) binding time (h)

24

740 741 742 743

Figure 4. Evaluation of competitive ligand exchange for Hg2+ in Hg(II)-DOM

744

complexes by CYS, GSH, DMPS, CTA, and EDTA, by stepwise determination of

745

HgR-AA, HgR-Sn, and HgNR. Complexes of EFPC-DOM-Hg(II) (a, c, e) and SHA-

746

Hg(II) (b, d, f) were formed by pre-equilibrating the DOM solutions with Hg2+ for either

747

1 or 24 h before the addition of competing ligands. The exchange reaction time was set

748

at 1 h after the addition of competing ligands. The initial Hg2+ concentration was 25

749

nM, and DOC:Hg molar ratios were 18400 and 19200 for EFPC-DOM and SHA,

750

respectively. All experiments were conducted in PBS (pH = 7.4). All data were

751

normalized to the total non-purgeable Hg(II) (HgNP).

752 753

31


A stepwise reduction approach reveals mercury competitive binding

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