Article pubs.acs.org/est
Cysteine Enhances Bioavailability of Copper to Marine Phytoplankton Michael J. Walsh,†,‡ Sarah D. Goodnow,† Grace E. Vezeau,†,§ Lubna V. Richter,† and Beth A. Ahner*,† †
Cornell University, Biological & Environmental Engineering, Ithaca, New York 14853-5701, United States S Supporting Information *
ABSTRACT: Emiliania huxleyi, a ubiquitous marine algae, was cultured under replete and Cu-limiting conditions to investigate Cu uptake strategies involving thiols and associated redox reactions; comparisons to a model diatom, Thalassiosira pseudonana, were also drawn. Cu-limitation increased rates of cell surface reduction of Cu(II) to Cu(I) in E. huxleyi but not in T. pseudonana. Furthermore, Cu-limited E. huxleyi cells took up more Cu when cysteine was present compared to when no ligand was added, although a dependence on cysteine concentration was not observed. In contrast, Cu uptake by replete cells was dependent upon the relative abundance of inorganic species [Cu(I)′]. We also show that cysteine can increase the bioavailability of Cu to Culimited cells, of both species, through the reductive release of Cu(I) from fairly strong Cu(II) ligands such as EDTA. Finally, support for a mechanism involving uptake of a Cys−Cu complex in E. huxleyi is drawn from the observation that Cu-limitation significantly enhances cysteine uptake by transporters that exhibit Michaelis−Menten kinetics. These Cu uptake strategies help explain the presence and distribution of dissolved thiols in surface seawater and have implications for the biogeochemical cycling of Cu in low Cu environments.
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INTRODUCTION
The chemical speciation of Cu(I) is distinct from that of Cu(II). Complexation of Cu(I) by thiols, such as glutathione or cysteine, is often assumed because thiols have a high affinity for Cu(I) and are present in seawater at concentrations similar to those of strong Cu(II) ligands.12,13 In fact, while the conditional stability constants are not that different from EDTA: log K′Cu(I)−Cys = 11,14 log K′Cu(I)−GSH = 12,14 log K′Cu(II)−EDTA = 11.8,15 the high chloride concentration of seawater drives equilibrium toward the formation of inorganic chloride−Cu(I) complexes (log K′CuCl = 3.1; log K′CuCl2 = 5.42; log K′CuCl3 = 4.7516). For example, if we assume a Cu(I) concentration of 0.2 nM (10% of total Cu)11 and 10 nM of a thiol or mix of thiols with binding strengths comparable to that of GSH,17 then approximately 90% of the Cu(I) would be complexed by chloride. A much stronger ligand, with a log K′ of 15, would be necessary to reduce Cu(I)′ concentrations to less than 1 pM or to predicted inorganic levels of Cu(II) from field electrochemical titrations of Cu2+.1−4 Studies have identified Cu(II) binding ligands with log K′ greater than or equal to 15,18 but the presence of strong Cu(I) complexes has only been postulated in the field and in specific cultures.19 Uptake of Cu by eukaryotic algae involves both low affinity (Figure 1-2) and high affinity (Figure 1-3) Cu uptake
Cu is an important micronutrient for microorganisms in surface seawater, where total copper concentrations are low (approximately 1−3 nM). The majority of studies focused on the chemical speciation of Cu in seawater have been performed using electrochemical techniques. These studies have shown that much of the Cu is bound to strong organic ligands which are typically present at higher concentrations.1−4 These ligands, presumed to be of biological origin, reduce the concentration of free cupric ion [Cu2+] and inorganic Cu(II) species [Cu(II)′] by several orders of magnitude (e.g., Bundy et al.:3 [Cu2+] < 1 fM). These results have led to speculation that there may be regions of the ocean in which copper levels are low enough to limit the growth of some algae species.2−4 However, one acknowledged limitation of these speciation studies is that there is no differentiation between Cu(I) and Cu(II).2,4 It is well established that a variable fraction of total dissolved Cu in surface seawater is present as Cu(I). Production of Cu(I) from Cu(II) occurs via photochemistry5 and via reduction by cell surface reductases.6 In an aerobic environment, Cu(I) is readily oxidized to Cu(II), but in seawater, Cu(I) is stabilized by inorganic ligands, such as chloride, which slows oxidation.7−9 Chemical processes alone predict the Cu(I) fraction to be between 0.1 and 4% of total copper,10 while measurements of steady-state Cu(I) concentrations in surface seawater range from 5 to 10% of total Cu concentrations.11 Greater percentages are measured in estuaries.9 © XXXX American Chemical Society
Received: April 27, 2015 Revised: September 8, 2015 Accepted: September 15, 2015
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DOI: 10.1021/acs.est.5b02112 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
of Cu from the cell.34 However, when in the presence of stronger ligands such as ethylenediaminetetraacetic acid (EDTA), glutathione and cysteine have been shown to enhance Zn bioavailability to E. huxleyi and Thalassiosira weissf logii,25 and thiols, in particular cysteine, have been observed to facilitate the uptake of metals such as Hg into bacteria that methylate Hg.35 As thiols appear to influence the uptake of various metals, are present in surface seawater, and can catalyze the reduction of Cu(II) to Cu(I),36 we performed a series of experiments to examine the influence of thiols on copper bioavailability to both Cu-replete and Cu-limited cells, with particular attention given to the redox state of Cu and biological and chemical drivers. Here, we propose a mechanism for thiol-mediated uptake of copper via the extracellular reductive release of EDTA-bound Cu(II) to Cu(I) by cysteine (Figure 1-C). We show that adding Cys to the culture medium increases the bioavailability of Cu bound to EDTA. While it is likely that the majority of the cysteine-reduced Cu(I) is assimilated through high-affinity Cu(I) transporters at the cell surface, we also provide circumstantial evidence for Cys−Cu(I) complex uptake through an independent transport mechanism. We demonstrate that components of these complementary uptake mechanisms are enhanced in E. huxleyi when the cells are Cu limited. Comparisons to Thalassiosira pseudonana were also made.
Figure 1. Mechanisms for copper reduction and uptake in surface seawater: Generally organic Cu(II)-binding ligands (L) prevent the uptake of copper by the cell, though lipophilic ligands may facilitate diffusion through the membrane (1).28 If Cu concentrations are high and/or not buffered by a ligand, then Cu2+ will be available for uptake via low affinity transporters (2).39 High affinity uptake (3) generally requires the generation of Cu(I) by cell surface reductases (A).6 Photochemically mediated production of Cu(I) (B) has been shown to occur in the bulk media.7 In this paper, we provide evidence for the enhancement of uptake via the cysteine-mediated reduction of Cu(II) to Cu(I) (C) that would supply Cu(I) for high affinity uptake (3), but we also explore the potential for cysteine-mediated uptake (4). Inorganic species, Cu(I)′ and Cu(II)′, are predominately complexes with chloride and, for Cu(II), with hydroxide.
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METHODS Culturing. Monocultures of E. huxleyi (CCMP1516) and T. pseudonana (CCMP1335) were obtained from the National Center for Marine Algae and Microbiota (NCMA, West Boothbay Harbor, ME, U.S.A.). Growth was monitored via fluorescence (Turner Instruments). Cultures were grown in acid-washed polycarbonate bottles under constant light (120 μmol photon m−2 s−1) at 20 °C using the synthetic ocean water medium Aquil,37 a trace metal defined medium. Trace metal ion concentrations were buffered with 100 μM EDTA (Fisher). Total added iron was reduced to 100 nM when culturing E. huxleyi, which is sufficient to maintain the maximal growth rate.38 The total Cu concentration for replete cultures was 20 nM (initial pCu2+= −log [Cu2+] = 13.8). Cu-limited cultures were prepared by transferring cells in mid-to-late exponential growth at least three times into fresh Aquil prepared with no added Cu, except when required to generate sufficient biomass following successive transfers. In this case, Cu was added to 1 nM, which is equivalent to estimated background Cu concentration39 (see ref 40 for cellular quotas and growth rates). Aquil with no trace metals, chelators, or vitamins (hereafter designated as synthetic ocean water or SOW) was used in cellular resuspensions and washes. Measurement of Chemically and Biologically Produced Cu(I). The colorimetric Cu(I) indicator bathocuproine disulfonate (BCS, limit of detection ∼1 μM Cu(I)) was used for detection of biologically generated Cu(I)6 and thiolproduced Cu(I) (as described using neocuproine36). Further analytical details are included in Figures 2, SI-1, SI-2, and SI-3 captions. To measure the biological reduction of Cu, 9 mL duplicates of concentrated resuspended algal culture (30 million cells mL−1 or ∼10-fold the biomass concentration prior to resuspension) were amended with 1 mM BCS, followed by variable additions of Cu(II). The volume was brought to 10 mL with SOW. Starting 5 min after the addition of Cu(II), duplicate samples were taken periodically over a period of 150 min and were immediately centrifuged at
systems.20 Generally, high affinity copper transporters, such as CTR1, require Cu(I),20 which can be generated by cell surface reductases (Figure 1A)6,21 or in the bulk medium through the photochemical reduction of Cu(II) (Figure 1B).5,22 These types of transporters rely on a chemical reaction of the metal ion with the cellular transport ligandthis reaction occurs much faster for the hydrated aqua-species and inorganic species compared to metals that are chelated by strong organic ligands.23 There is evidence that Cu in organic complexes can become bioavailable,24 and several possible mechanisms have been evoked to explain this phenomenon including dissociation of organic complexes at the cell surface, ternary complex formation,25 and reductive dissociation of strong organic complexes, as has been proposed to occur with Fe.26,27 Alternative mechanisms in which Cu ligands may in fact facilitate uptake have also been investigated. For example, Cu accumulation can be enhanced by the addition of ligands that form lipophilic complexes which diffuse through cell membranes (Figure 1-1)28 or by the addition of cell exudates via an unknown mechanism.29 Ligand-enhanced uptake has also been observed in field experiments.30 Thiol ligands, in particular, have been postulated to be involved in Cu uptake,30,31 but are more often studied in the context of detoxification and efflux because marine algae produce and exude thiols, such as glutathione (e.g., ref 32), under high copper stress. For example, when exposed to elevated copper concentrations, Emiliania huxleyi accumulates elevated levels of both cysteine and two cysteine-containing dipeptides,33 both of which appear to be involved in the export B
DOI: 10.1021/acs.est.5b02112 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
equilibrated with EDTA or oxidized glutathione (GSSG, Sigma) were made to cell resuspensions which were incubated for 60 min in the growth chamber under the same conditions used for cultivation. Cells were then harvested for Cu measurements. Speciation of copper in SOW was calculated with the software package VisualMINTEQ,42 using stability constants for cysteine and GSH.14 Cu uptake was also measured in experiments with resuspended cells to which an initial addition of Cu−EDTA (100 nM Cu and 1000 nM EDTA) was made. Samples were taken between 30 and 60 min after the addition of Cu−EDTA. The culture was then divided for the addition of either Dcysteine, L-cysteine or GSH. Controls received no addition. After 60 more minutes of incubation in the growth chamber, triplicate samples were taken from each treatment. In all biological Cu uptake experiments, an initial sample was taken after cell resuspension, before the addition of any copper or ligand. Cu uptake is reported as the difference between cellular Cu after treatment and this initial amount. All samples were made in triplicate. Cell filtration, washing, acid digestion, and measurement via ICP-MS are described in Walsh and Ahner.40 Measurement of Cysteine Uptake. [35S]Cysteine (Lisomer, GE Life Sciences) was diluted to a specific activity of 1000 Ci mol−1, separated into stock solutions and stored at −20 °C. Immediately before use, stock solutions were thawed and added to 50 μM unlabeled Cys to obtain a specific activity of 100 Ci mol−1. Cu-replete and Cu-limited cells were resuspended in SOW to which additions of [35S]Cys were made at concentrations ranging from 10 to 500 nM. From 0 to 30 min, at 5 min intervals, 20 mL aliquots were removed from each treatment and filtered onto duplicate GF/A filters. To terminate [35S]Cys uptake, filters were washed with 5 mL of 1 mM unlabeled cysteine in ice cold SOW, followed by 10 mL ice cold SOW. Cell-associated [35S]Cys was measured by liquid scintillation counting (Beckman LS6500).
Figure 2. Measurements of Cu(I) production rates in the presence of replete (■) and Cu-limited (□) E. huxleyi cells at several micromolar concentrations of added Cu(II). Rates were determined from a linear fit of Cu(I)−BCS generation (λmax = 483 nm; εmax = 12 700; BioTek Synergy4Microplate Reader Spectrophotometer) measured at 5 min intervals for an hour, only the linear portion of the time course was used. Total Cu(I) generated ranged from 5% (replete) and 10% (Culimited) of total added Cu(II) at 400 μM addition to approximately 30% (replete) and 40% (Cu-limited) at 50 μM. Lines represent nonlinear least-squares fits to the Michaelis−Menten equation (replete: Vmax = 1.07 ± 0.26 fmol cell −1 hr−1, Km = 123 ± 21 μM; Cu-limited: Vmax = 2.43 ± 0.23 fmol cell −1 hr−1, Km = 123 ± 32 μM).
1000 × g for 5 min. Aliquots of each cell-free supernatant (200 μL) were pipetted into a 96-well plate. Note that BCS also serves as a weak Cu(II) ligand, increasing Cu(II) solubility and enabling experimentation at higher Cu(II) concentrations. Measurements of Cu(I)−BCS formation were made in cell-free spent media to determine background reduction by dissolved constituents and subtracted from those made in the presence of cells. Rates are reported on a per cell basis because the surface area of the two algae species are similar.40 Generation of Cu(I) for Uptake Experiments. For uptake experiments involving Cu(I), a 1 mM stock solution of Cu(I) was generated from Cu(II)O via gaseous reduction with SO2(g).14,41 When Cu(I) was added with a ligand, components were pre-equilibrated together in 5 mL of N2sparged (Airgas) SOW for 15 min before addition. BCS colorimetric measurements of Cu(I) confirmed concentrations prior to addition. Biological Uptake Experiments. Copper uptake experiments were performed with resuspended algal cultures, using trace metal clean techniques to minimize Cu contamination and a cell-surface wash that included diethylene triamine pentaacetic acid (DTPA, Fisher) and dithiothreitol (DTT, Sigma) to remove adsorbed Cu.40 In experiments testing the effect of different ligands on Cu uptake, cells were harvested by centrifugation as described in ref 40. In experiments testing the addition of Cu(II)−EDTA and cysteine, cells were filtered onto acid-washed polycarbonate filters (0.8 μm) with low vacuum pressure (
