Article pubs.acs.org/est
Weak Organic Ligands Enhance Zinc Uptake in Marine Phytoplankton Ludmilla Aristilde,‡,§ Yan Xu,*,‡ and François M. M. Morel Department of Geosciences, Princeton University, Princeton, New Jersey 08540, United States S Supporting Information *
ABSTRACT: A recent study of the effect of pH on Zn and Cd bioavailability shows that binding to weak organic ligands can increase the pool of metals available to phytoplankton in the presence of strong chelating agents. We explore the underlying mechanism in laboratory experiments with the model species Emiliania huxleyi and Thalassiosira weissf logii. Additions of L- and D- isomers of cysteine (Cys) result in similar increases in Zn uptake rates in the presence of the strong chelator ethylenediaminetetraacetic acid (EDTA) but decrease it in the absence of EDTA, ruling out uptake by a specific Zn−Cys transporter. The effect of Cys does not result from alleviating diffusion limitation of inorganic Zn. The enhancement of Zn uptake kinetics by weak ligands is consistent with a mechanism involving formation of a transient ternary complex with uptake molecules: (1) the enhancement is most dramatic in Zn limited cells whose high affinity transporters should be most effective at extracting Zn from weak ligands; (2) the enhancement occurs with a variety of weak ligands, demonstrating that the underlying mechanism has little chemical specificity; and (3) no enhancement of uptake is seen when Zn is bound in complexes that would make formation of multiligand complexes with uptake molecules difficult. Weak complexing agents which have received heretofore little attention may play a key role in the bioavailability of metals in natural waters.
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INTRODUCTION In surface seawater, most biologically essential metals such as Fe, Zn, or Cu are bound to strong chelating ligands whose identity is generally unknown.1−3 Numerous laboratory studies, using chiefly the artificial strong chelating agent ethylenediaminetetraacetic acid (EDTA), have shown that the unchelated metal concentration controls the kinetics of metal uptake by phytoplankton (for example, ref 4). In other words, in the presence of EDTA, the bioavailability of a given metal, M, is determined by the sum, M′, of the concentrations of the metal complexes with the major inorganic ligands of seawater (principally HCO3−, CO32‑, Cl−, SO42‑, in addition to OH− and H2O), a pool which we refer to as “inorganic M” (in accord with tradition in oceanography but contrary to terminology in bioinorganic chemistry). But, binding to EDTA may not mimic adequately the effect of binding to natural chelators and the inorganic concentration M′ may not adequately represent the bioavailable concentration of a metal in surface seawater. This is obviously true when microorganisms can take up a metal bound by a strong metal chelating agent. A well-known example is that of Fe which can be taken up by some microorganisms when chelated with siderophores, either by transport of the chelate itself5−7 or via reduction of the bound Fe(III) and uptake of Fe(II).8 A more general reason why the inorganic concentration M′ may not represent the bioavailable concentration in the field is the presence, in surface seawater, of a number of weak, and generally uncharacterized, ligands.2,7,9 In a recent study,10 we showed that, in the presence of EDTA, addition of weak © 2012 American Chemical Society
complexing agents could increase the rate of uptake of Zn and Cd by model phytoplankton species. When an organic ligand, L, is added to a medium containing a metal M bound only to inorganic ligands (M′ = MT), M binds to L, and the inorganic concentration M′ decreases stoichiometrically as the concentration of L increases (Figure 1A). But when the inorganic concentration of a metal, M′, is buffered by binding to a strong chelating agent in excess, Y, only a small amount of M becomes bound to L, while the bulk of M remains bound in the chelate MY (Figure 1B). If the concentration of ML is small compared to that of the total metal MT (≈ [MY]), the resulting inorganic concentration M′ is negligibly smaller than its original value in the absence of L. Nonetheless, the concentration of the weak complex ML can be much larger than the inorganic concentration M′ (Figure 1B). We use the terminology “weak” and “strong” ligands or complexes for convenience, but the differentiation between the two is not simply dependent upon the effective affinities of the ligands for the metal (for example, in seawater EDTA and Cys have nearly the same effective affinity for Zn2+) but also, as is made clear later, on the lability of the complexes (the rate of dissociation of ZnEDTA being, for example, much slower than the rate of dissociation of ZnCys). Received: Revised: Accepted: Published: 5438
January 26, 2012 April 5, 2012 April 12, 2012 April 12, 2012 dx.doi.org/10.1021/es300335u | Environ. Sci. Technol. 2012, 46, 5438−5445
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Figure 1. (A) Concentrations of the total free Zn, Zn′, and ZnCys complexes as a function of Cys concentration. ([Zn]tot = 2 nM, pH 8.1). (B) Concentrations of Zn′, ZnCys, and ZnEDTA complexes as a function of Cys concentration. ([Zn]tot = 50 nM, [Cys]tot = 2 μM, [EDTA]tot = 100 μM, pH 8.1). (C) Schematic illustration of mechanisms by which addition of a weak ligand (L) can increase the uptake rate of a metal (M) by phytoplankton; M complexed to strong ligand (M−Y), M complexed to weak ligand (M−L), inorganic M in the bulk solution (M′b), inorganic M at the cell surface (M′s). (D) Structures of the L ligands used in this study: cysteine (Cys), glutathione (GSH), phytochelatin-2 (PC2), histidine (His), and desferrioxamine-B (DFB).
As noted previously11,12 and illustrated in Figure 1C, there are, in principle, three mechanisms by which complexation by a weak ligand can increase the rate of uptake of M by a phytoplankton cell (or indeed any microorganism): (1) the complex ML may be transported inside the cell; (2) the dissociation of ML may increase the inorganic metal concentration in the vicinity of the cell; or (3) the complex ML may be available for uptake without internalization of ML; this can occur if ML delivers M to a metal uptake molecule, X, by forming a transient ternary complex XML, thus transferring M directly to the cell for uptake. Mechanism 1 is straightforward and is identical in its principle to the uptake of Fe-siderophore complexes by bacteria and fungi and of a few complexes of other metals with “metallophores” (refs 5, 6, 13 and references therein). Enhanced uptake of trace metals has been observed in the presence of some organic compounds that are weak complexing agents. This has been seen with lipophilic compounds that diffuse readily through lipid membranes (for example, 14−16). Metal uptake may also occur via the transport system for substrate compounds that are metabolized by cells. For example the uptake of Cd2+ by the green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was increased in the presence of citrate, and this result was ascribed to “piggyback uptake” of the Cd−citrate complex by the citrate transport system.17
An increase in uptake rate through mechanism 2 can occur whenever the cellular uptake rate of M becomes limited by the diffusive flux of inorganic M in the boundary layer of the cell. In that situation the concentration of inorganic metal at the surface of the cell, M′S, which results from a balance between the diffusive supply of inorganic metal in the cell boundary layer and its uptake by the cell, is lower than the value M′b in the bulk solution. Depending on the bulk concentration of ML, its diffusive flux in the boundary layer can be large and its dissociation can increase the value of M′S and, hence the uptake rate of M. For example, Hassler and Wilkinson have shown that the Zn uptake rate in Chlorella kesslerii exceeded the predicted diffusion limitation of free Zn ion in the presence of nitrilotriacetic acid (NTA).18 An increase in metal uptake upon addition of complexing agents has also been observed in plant roots under condition of diffusion limitation and been attributed to complex dissociation in the diffusion boundary layer.19−21 A similar situation is encountered in amperometric measurements where the concentration of a free metal that is reducible at an electrode surface can be increased by dissociation of complexes in the boundary layer at the surface of the electrode (for example, 22). Mechanism 3 is in some ways similar to mechanism 1 insofar as it results in the complex ML being effectively bioavailable. But contrary to mechanism 1, mechanism 3 does not require that the weak ligand L be hydrophobic or that it be transported 5439
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were grown at 20 °C under continuous light (80−100 μmol quanta m−2 s−1). Short-Term Uptake of Zn. The uptake media were prepared using 0.2-μm filtered Gulf Stream seawater, which contains about 80 μM DOC.29 The pH of seawater was manipulated by adding ultrapure HCl and NaOH and measured by potentiometer. The seawater was equilibrated overnight with 100 μM EDTA before adding Zn and the other ligands (L-Cys, D-Cys, L-GSH, and L-His, PC-2, or DFB). For experiments in the presence of these ligands, the ligand was equilibrated first with Zn in a small volume of water at about pH 8 for 2−4 h and then the complex was added into the EDTA-buffered seawater to equilibrate for an additional 2−4 h before adding cells. TCEP (tris-2-carboxyethyl-phosphate) was also added as the reducing agent for Cys, GSH, and PC-2; experiments were performed to ascertain that the presence of TCEP did not cause any appreciable difference in uptake compared to no TCEP. The following Zn uptake experiments were conducted in the EDTA-buffered seawater with weak ligands equilibrated with 50 nM Zntot (control experiments without any added weak ligand were performed in each case): uptake by Zn-limited E. huxleyi and T. weissf logii cells with L-Cys (2 μM), D-Cys (2 μM), LGSH (22 μM), PC-2 (1 μM), L-His (40 μM), or DFB (8 μM) at pH 8.1; uptake by Zn-replete E. huxleyi and T. weissf logii with L-Cys (2 μM), L-GSH (22 μM), or L-His (40 μM) at pH 8.1; uptake by both Zn-limited and Zn-replete E. huxleyi and T. weissf logii cells at various [L-Cys] (0.5, 0.8, 5, 20, or 40 μM); and, uptake by Zn-limited T. weissf logii cells with 2 μM and 20 μM L-Cys at pH 7.9. To assess the Zn transport capacity of the T. weissf logii cells, uptake experiments were conducted in the EDTA-buffered seawater as a function of increasing [Zn]tot at pH 8.1: 0, 10, 50, 100, 200, 500, 1050, 4000, and 10 000 nM with Zn-replete cells and 0, 10.5, 31.1, 48, 129.5, 150, 286.9, 390, 750, 1000, 1055, 7500, and 10 010 nM with Zn-limited cells. For the uptake experiment in the absence of EDTA, Zn was first equilibrated with L-Cys in small volume of water at about pH 7 for 3 h and then the complex was added into seawater to equilibrate for another hour before adding the cells. Control treatments were conducted in the same way but without addition of Cys. Calculations of the Zn speciation in each experiment were computed using MINEQL+,30 published stability constants,31 and taking into account the salt and ligand concentrations, and pH and alkalinity conditions. All uptake measurements were obtained from up to four independent experiments (i.e., biological replicates with newly grown cells), with each single experiment conducted in duplicate. To quantify the cell-normalized uptake rate, the cell concentration in each bottle was measured using a Beckman Coulter Counter: 10 000−50 000 cells mL−1 for T. weissf logii (counting parameter: 7 to 21 μm) and 50 000−100 000 cells mL−1 for E. huxleyi (counting parameter: 2.8 to 9 μM). To interpret the data quantitatively, the total Zn concentration, [Zn]tot, was taken as the concentration added plus 3.5 nM (the background concentration in Sargasso seawater plus contamination from handling of seawater and bottles, measured by voltammetry, O. Baars, personal communication). Because both the calculation of Zn uptake rate from tracer experiments and the concentrations of Zn′ and ZnCys vary linearly with [Zn]tot, our conclusions are unaffected by the exact value of the background Zn concentration. Because the strong ligand in our Sargasso seawater is only 1.24 nM
by a specific transport system. Formation of a transient ternary complex is a common mechanism that accelerates the metal transfer between ligands (ligand exchange).12,23,24 The internalization of M into the cell from weak complexes ML can occur whenever a ternary complex XML forms with metal uptake ligands X. This uptake mechanism should thus be relatively promiscuous rather than specific for a particular compound. Uptake of a metal via mechanism 3 should be essentially invisible in the absence of a strong complexing agent that buffers the value of M′. In this case, the addition of a weak complexing agent L normally decreases the uptake rate of M because the rate of uptake of M from ML (or XML) is typically slower than the rate of uptake of the inorganic metal whose concentration decreases stoichiometrically with the formation of ML (Figure 1A). Therefore, to obtain an increase in uptake rate through this third mechanism, the decrease in M′ caused by the addition of L must be small enough that the uptake of M from ML more than makes up for the reduced rate of uptake of M′. This condition is met when M′ is buffered by a strong chelating agent and practically unaffected by the addition of L (Figure 1B). Here, we explore the aforementioned mechanisms by examining the uptake of Zn, in the presence of both the strong ligand EDTA and the weak ligand cysteine (Cys), by two model marine phytoplankton, the diatom Thalassiosira weissf logii and the coccolithophore Emiliania huxleyi. This should be a convenient model system since, in a previous study on the effect of pH on Zn uptake,10 we have observed that Cys enhances Zn uptake by T. weissf logii in the presence of EDTA. Available thermodynamic data for the formation of Zn−Cys complexes and published studies on the kinetics of Zn uptake by these model organisms in the presence of EDTA only (when Zn uptake depends on Zn′) provide a basis for interpreting our results quantitatively. We also compare our results with Cys to those obtained with other ligands: glutathione (GSH), histidine (His), phytochelatin [PC-2 = γ(Glu−Cys) 2 −Gly] and desferriferioxamine B (DFB) (Figure 1D). Thiol-containing ligands, including Cys and GSH, are known to be released in the growth medium of E. huxleyi, T. weissf logii and other marine microalgae,25−28 such that, in addition to exemplifying a general mechanism, our study may be directly relevant to natural conditions.
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EXPERIMENTAL SECTION Cultures and Chemicals. E. huxleyi strain PLY 92E was obtained from the Plymouth Culture Collection of Marine Algae in the UK and T. weissf logii CCMP1336 was obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton in Maine. All chemicals were obtained analytical grade from Fisher or Sigma-Aldrich except for phytochelatin (AnaSpec). Culturing. Bottles used for all culturing and uptake experiments were acid-cleaned polycarbonate bottles. Culture media were prepared using 0.2-μm filtered and microwavesterilized Gulf Stream seawater enriched with chelexed and filter-sterilized macronutrients and filter-sterilized trace metals (20 nM Cu, 120 nM Mn, 10 nM Se) buffered with 100 μM EDTA and vitamins. The culturing solutions contained 100 μM NO3−, 10 μM PO43−, 100 μM SiO2, and 1 μM Fe for T. weissf logii, and 50 μM NO3−, 2 μM PO43−, and 87 nM Fe for E. huxleyi. For Zn-replete cells, 98 nM Zn was added, and for Zn limited T. weissf logii or E. huxleyi, 29 nM and 6.45 nM Zn were added, respectively. Cells for cultures and short-term uptake 5440
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Figure 2. Short-term Zn uptake rate by Zn-limited E. huxleyi (A) and T. weissf logii (B and C). In A and B, control: [EDTA]tot = 100 μM and [Zn]tot = 50 nM; L-Cys and D-Cys: [Cys]tot = 2 μM, [EDTA]tot = 100 μM and [Zn]tot = 50 nM; pH 8.1. In C, control: [Zn]tot = 4.5 nM; L-Cys: [Cys]tot = 2 μM and [Zn]tot = 4.5 nM; pH 8.2.
that uptake of the inorganic metal be limited by diffusion. The diffusion flux, J, of the inorganic metal to the cell is given by
(measured by voltammetry, O. Baars, personal communication), the bulk of the Zn is Zn′ in the control treatment. In each experiment, exponentially growing cells were filtered onto acid-cleaned polycarbonate membrane filters, rinsed five times with 0.2-μm filtered Gulf Stream seawater and resuspended in seawater. Aliquots were then dispensed into the uptake media. The radiotracer 65Zn was added as ZnCl2 in 10 mM HCl to measure the Zn uptake for 2−4 h. The concentration of Zn in 65Zn stock solution was reported by the provider (Oak Ridge National Laboratory) and confirmed by spectrophotometry using 4-(pyridyl-2-azo)resorcinol (PAR). Because only a few microliters of 65Zn stock solution was used in each experiment, the pH of the uptake medium was not affected by the addition. At intervals of 0.5 to 1 h, 20-mL aliquots from each bottle were removed and filtered onto polycarbonate membrane filters; 3-μm filters were used for T. weissf logii and 1-μm filters were used for E. huxleyi. To remove Zn bound to the outside of cells, the filters were washed with an oxalate−EDTA solution for 5 min32,33 and 65Zn retained on the membrane was measured via liquid scintillation counting.
J = 4πRD(Zn′b − Zn′s )
(1)
where R is the effective radius of the cell (2.5 μm for E. huxleyi and 5.6 μm for T. weissf logii), D is the diffusion coefficient for inorganic Zn (2.16 × 10−2 cm2 h−1),34 and Zn′b and Zn′s are the Zn′ values in the bulk solution and at the cell surface, respectively. The likelihood that diffusion may limit uptake of the inorganic metal is given by a simple comparison of the measured uptake rate in the absence of Cys to the maximum diffusion flux, Jmax, of the inorganic metal to the cell surface (Zn′s = 0). The values of Jmax calculated for the conditions of Figure 3 (Zn′b = 13.3 pM) are much larger than the uptake rates measured in the absence of Cys, particularly in the case of E. huxleyi (Figure 3). Enhancement of uptake by increasing the diffusive flux to the surface of the cells according to mechanism 2 thus seems unlikely.
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RESULTS AND DISCUSSION We have shown previously10 that the addition of Cys to a medium where Zn is buffered by an excess of EDTA results in an increase in Zn uptake by Zn-limited T. weissf logii cells. We repeated these experiments with Zn-limited cells of both E. huxleyi and T. weissf logii and observed Cys-enhanced uptake in both organisms, showing that the phenomenon is not limited to a particular organism or to diatoms (Figure 2A and 2B). Mechanism 1: Uptake of ML Complex. The enhancement of Zn uptake in the presence of Cys occurs whether the Dor L-stereoisomer of Cys is added (Figures 2A and 2B), making it very doubtful that a Zn−Cys complex is the substrate of a specific uptake system. This conclusion is confirmed by uptake experiments in the absence of EDTA. Addition of Cys to a medium where Zn′ is not buffered by EDTA results in a decrease rather than an increase in uptake rate (Figure 2C), providing prima facie evidence that the Zn−Cys complexes are not taken up directly, or, at least, not efficiently, by the cell. We note that this result also rules out the unlikely possibility that the enhancement of Zn uptake by Cys addition in the presence of EDTA results from a direct physiological effect of Cys on the cells rather than a change in Zn speciation in the medium. However, the quantitative interpretation of these experiments is subtle as discussed below. Mechanism 2: Enhancement of Diffusion-Limited Uptake. Enhancement of Zn uptake by mechanism 2 requires
Figure 3. Short-term Zn uptake rate by Zn-limited (black bars) and Zn-replete (white bars) E. huxleyi (A) and T. weissf logii (B). Control: measured uptake rate at 100 μM EDTAtot and 50 nM Zntot; Max enhanct: maximum enhancement (measured uptake rate times Zn′b/ Zn′s); +Cys: measured uptake rate at 2 μM Cystot, 100 μM EDTAtot and 50 nM Zntot. Dash lines: calculated maximum Zn′ diffusion rate. 5441
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Figure 4. Short-term Zn uptake rate by Zn-limited (filled symbols) and Zn-replete (open symbols) E. huxleyi (A) and T. weissf logii (B−E). In A, B, and D, the uptake rates were measured as a function of Cys concentration at constant [EDTA]tot (100 μM) and [Zn]tot (50 nM). In C and E, the uptake rates were measured in the absence of Cys at varying [Zn]tot and constant [EDTA]tot (100 μM); the Zn concentration is reported as the total inorganic species of Zn, Zn′.
“extracting” Zn from Cys than under Zn-replete conditions. We explored this question further by performing uptake experiments under a range of Cys concentrations (0−40 uM) in the presence of 50 nM Zntot and 100 uM EDTA. As seen in Figure 1, under these conditions, Zn′ is buffered at 13.3 pM. The Zn uptake rate by E. huxleyi and T. weissf logii increased with the concentrations of Cys and Zn−Cys complexes under all conditions (Figure 4) but this increase was much smaller and plateaued out at lower Zn−Cys concentrations in Zn-replete than in Zn-limited cells. Moreover, a comparison of the maximum Zn uptake rates observed at high Zn−Cys concentration and at high Zn′ in the absence of Cys shows contrasting results for Zn-replete and Zn-limited T. weissf logii cells. In Zn-replete cells, the Zn uptake rate achieved at high Zn−Cys concentration is only a small fraction of the one at high Zn′ (Figure 4B and 4C), confirming that the low affinity Zn transporters are inefficient at taking up Zn from Zn−Cys complexes. In contrast, in Zn-limited cells, the Zn uptake rate achieved at high Cys concentration is nearly the same as the one at high Zn′ (Figure 4D and 4E), showing that the high affinity Zn transporters are about as efficient at taking up Zn from Zn−Cys complexes as from the inorganic Zn pool. Although the nature of the Zn transporters in T. weissf logii and E. huxleyi has not been elucidated, they are likely to be related to the Zrt/Irt-like proteins (ZIP) and heavy metal P-type ATPases (HMAs),36−38 which have homologues in the T. pseudonana and E. huxleyi genomes. The greater ability of Znlimited cells to take up Zn from Zn−ligand complexes may for example correspond to a switch from the histidine-rich binding site in ZIP proteins to the cysteine-rich binding site in HMA. Enhancement of uptake by mechanism 3 should occur with a wide range of weak ligands, L, as long as a ternary complex XZnL can readily be formed with the cellular transporters X at the cell surface. We tested the effect of adding a variety of
A more precise calculation of the maximum enhancement that could result from mechanism 2 is obtained by comparing the bulk inorganic metal concentration Zn′b to the concentration at the cell surface Zn′s resulting from the steady state between diffusion of the inorganic metal from the bulk and uptake by the cell (obtained by replacing J by the measured uptake rate and Zn′b by its value in eq 1). Dissociation of Zn− Cys complexes could increase Zn′s to Zn′b, at the most, so that the maximum possible enhancement of uptake is given by the ratio Zn′b/ Zn′s. As seen in Figure 3, the maximum enhanced uptake rates of the inorganic metal calculated in this way are much smaller than the uptake rate measured in the presence of Cys. So even a complete elimination of diffusion limitation is insufficient to account for the enhancement of Zn uptake rate by Cys measured in both E. huxleyi and T. weissf logii under either Zn-replete or -limited conditions. Calculations made with different sets of parameters (notably different stability constants for the Zn−EDTA complexes) do not modify this conclusion. Mechanism 3: Ternary Complex with a Transporter at the Cell Surface. Neither mechanism 1 nor mechanism 2 explain the experimental data. Mechanism 3, which involves the uptake of Zn via formation of a ternary complex with a metal uptake molecule, must thus be responsible for the observed enhancement of Zn uptake in the presence of the various weak ligands. There is no obvious direct experimental test to demonstrate this mechanism but the observed Zn uptake kinetics over a range of conditions must be consistent with the hypothesized underlying chemical process. From the data of Figure 3, it appears that the enhancement of Zn uptake by addition of Cys is markedly greater in Znlimited than in Zn-replete cells. This is consistent with mechanism 3 since the Zn transporters at the cell surface are known to increase in both quantity and in affinity under Znlimited conditions4,35 and they should be more effective at 5442
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ligands on Zn uptake by E. huxleyi and T. weissf logii from an EDTA-buffered medium (Figure 5). The large enhancement of
Figure 6. Cys-enhanced Zn uptake rate (= measured uptake rate − uptake rate due to Zn′) as a function of [ZnCys] (A) or [Zn(Cys)2] (B). Filled symbols: 100 μM EDTAtot, 50 nM Zntot, and various amount of Cys at two different pH values (7.9 and 8.1). Open symbols: no EDTA, 4.5 nM Zntot, and 50 μM Cystot at pH 7.9 and 8.2.
Figure 5. Short-term Zn uptake rate by Zn-limited E. huxleyi (A) and T. weissflogii (B) cells in the absence (control) and presence of weak ligands: L-cysteine (L-Cys), L-glutathione (L-GSH), phytochelatin (PC2), L-histidine (L-His), or desferrioxamine-B (DFB). Experimental conditions: [EDTA]tot = 100 μM, [Zn]tot= 50 nM, [L-Cys]tot = 2 μM, [L-GSH]tot = 22 μM, [PC2]tot = 1 μM, [L-His]tot = 40 μM, [DFB]tot = 8 μM, pH = 8.1. The concentrations of the different ligands were chosen in order to obtain [Zn-ligand]tot ≈ [Zn-Cys]tot at [Cys]tot = 2 μM.
single straight line when plotted as a function of [ZnCys], but not when plotted as a function of [Zn(Cys)2]. In particular, the experiments performed in the absence of EDTA (open symbols) yield uptake rates similar to those in the presence of EDTA for the same concentration of ZnCys but not for the same concentration of Zn(Cys)2. We conclude that the 1:1 Zn−Cys complex is the primary substrate for Zn uptake, consistent with the fact this complex leaves free coordination sites on the metal for facile formation of a ternary complex with uptake molecules. Our results clearly demonstrate that weak ligands can enhance the bioavailability of trace metals to phytoplankton in the presence of strong chelating agents. In view of the apparent lack of specificity of the underlying mechanism, we expect that this effect is quite general and occurs for several essential metals and many microorganisms in the presence of many types of ligands. For example, the uptake of a metal such as Cd2+, which forms relatively stable complexes with inorganic ligands in seawater (CdCl+, CdCl20), may occur through formation of ternary complexes (XCdClx) with uptake ligands. Such a mechanism may represent a particularly effective evolutionary strategy for organisms that must thrive in an environment where essential metals are both extremely rare and bound to strong chelators. In surface seawater, the binding of essential metals to chelating agentsthe sine qua non condition for the beneficial effect of weak ligandshas been demonstrated with electrochemical measurements.1−3,40−42 We do not know, however, whether the weak ligands that are present form metal complexes that are sufficiently strong to bind a significant fraction of the metals and sufficiently labile to transfer the metals to uptake molecules on cell surfaces. Our previous study of the pH effect on Zn uptake in natural samples from the New Jersey coast and the Gulf of Alaska indicates that such ligands may exist.10 Additional experiments with natural seawater samples are needed to establish the extent of the importance of the uptake of weak metal complexes by marine phytoplankton.
uptake observed with the addition of GSH, PC-2, and His, in addition to L- and D-Cys demonstrate that, as expected, the observed effect has little chemical specificity (Figure 2 and 5). Only with the addition of DFB, was there no significant enhancement in Zn uptake rate in either phytoplankton species, compared to the control with EDTA only (Figure 5). DFB is a trihydroxamate siderophore produced by bacteria with relatively modest affinity for Zn2+. But DFB is known to bind to Zn2+ at 5 or 6 coordination sites.39 As a result the formation of a ternary complex with uptake ligands should be difficult, consistent with the ineffectiveness of DFB to enhance Zn uptake in our experiments (Figure 5). A quantitative analysis of Zn uptake kinetics in the presence of cysteine provides further support to the idea that this uptake depends on the formation of a ternary complex at the cell surface. The complexation of Zn by Cys yields both a 1:1 and a 1:2 complex, ZnCys and Zn(Cys)2. In our previous study,10 we observed that the Zn uptake rate appeared proportional to the 1:1 but not the 1:2 complex and proposed that ZnCys but not Zn(Cys)2 served as substrate for uptake. This would be consistent with the fact that a ternary XZnCys complex should more readily form than a quaternary XZn(Cys)2 complex. The ratio of the 1:1 to the 1:2 complex varies with both the concentration of Cys and the pH. To test the relative roles of ZnCys and Zn(Cys)2 as substrates for uptake we analyzed quantitatively all our experiments performed at varying Cys concentrations and pH (Table S1). As seen in Figure 6, the Cys-enhanced uptake rate (defined as the measured uptake rate minus the calculated inorganic metal uptake rate) follows a 5443
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ASSOCIATED CONTENT
S Supporting Information *
Table S1. Summary of the short-term Zn uptake rate at various [Cys]tot and different pH values. This material is available free of charge via the Internet at http://pubs.acs.org
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AUTHOR INFORMATION
Corresponding Author
*Phone: (609) 258-1052; fax: (609) 258-5242; e-mail: yxu@ princeton.edu. Present Address §
Institute of Integrative Genomics, Carl Icahn Laboratory, Princeton University, Princeton, NJ 08540. Author Contributions ‡
These authors contributed equally to the work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Ja-Myung Kim for help in performing uptake experiments and Oliver Baars for measurements of Zn and ligand concentrations in seawater. This work was supported by grants from the National Science Foundation. L.A. acknowledges a postdoctoral fellowship from the National Science Foundation.
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