Article pubs.acs.org/IC
Unexpected Interactions of the Cyanobacterial Metallothionein SmtA with Uranium Celin Acharya† and Claudia A. Blindauer*,§ †
Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India Department of Chemistry, University of Warwick, Coventry, United Kingdom
§
S Supporting Information *
ABSTRACT: Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)nZn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. 1H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu+, Zn2+, Cd2+, Hg2+, and Pb2+ and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bioremediation.
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tion, including Cupriavidus metallidurans,11 Rhodopseudomonas palustris,11 Bacillus sphaericus,12 Deinococcus radiodurans,13 and Geobacter sulf urreducens.14 Immobilization of uranium by bacteria may involve several different processes, including biosorption of UO22+ at the cell surface, dissimilatory reduction of U(VI) to U(IV), with subsequent precipitation of insoluble UO2 or U(IV) phosphates or carbonates. However, much less is known about intracellular effects and pathways of uranium. To understand cellular effects of uranium, and also to be able to bioengineer bacterial strains with superior tolerance toward and/or accumulation of uranium, it is important to consider intracellular speciation, toxicity pathways, and mechanisms for detoxification. In this context, there has been increasing interest in studying the interactions of uranyl with proteins, either with selected candidates10,15 or in the context of metalloproteomics studies.16−21 In a study on the model uranium accumulator Procambarus clarkii, a freshwater crayfish, uranium was detected in fractions containing lowmolecular weight proteins, designated as “MT-like fraction”.19 “MT” stands for metallothioneins, an important class of mostly intracellular proteins with roles in both heavy metal detoxification and reactive oxygen species scavenging.22−26 They are stress-response metalloproteins, characterized by their
INTRODUCTION Interactions between bacteria and heavy metals are an intensely studied research area,1 with major applications in bioremediation.2−5 Moreover, these interactions are also important for understanding the roles that microbes have played in biomineralization over geological time scales.6 These considerations also hold for uranium, the key element for energy production through nuclear fission. In ores, it predominantly occurs as insoluble UO2 (uraninite), whereas in aqueous solution, the linear uranyl dication UO22+ dominates, with a varying number of further ligands (H2O/OH−, carbonate, other anions) in the equatorial plane around the U(VI) center. At natural abundance, uranium exhibits both radiotoxicity (primarily from 235U) and chemical toxicity, with the latter being also of importance for depleted uranium (DU, largely consisting of 238U), a byproduct of 235U enrichment, that is employed in armor and ammunition.7 The chemical toxicity of dissolved uranium is of considerable environmental significance and poses a major concern for public health and safety. Acute and chronic toxicological effects8 include kidney damage9 and impairment of bone formation.10 The significant anthropogenic mobilization of uranium through mining activities and nuclear fuel reprocessing has resulted in contaminated soil, sediments, and water. Several bacterial strains have been isolated from such contaminated sites and are being investigated in the context of bioremedia© XXXX American Chemical Society
Received: October 8, 2015
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DOI: 10.1021/acs.inorgchem.5b02327 Inorg. Chem. XXXX, XXX, XXX−XXX
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(SmtB).61 Recombinantly expressed SmtA can be isolated with four Zn2+ bound to the purified protein; its structure contains a single M4Cys9His2 cluster (Figure 1).62
small size (usually less than 10 kDa) and high metal and sulfur contents. MTs bind strongly to soft monovalent group 11 (Cu+) and divalent group 12 (Zn2+, Cd2+) metal ions both in vitro and in vivo.27,28 The cysteinyl thiols (-SH) of MTs complex their cognate metal ions in polynuclear metal−thiolate clusters, usually giving rise to ordered protein structures, whereas metal-free forms (apo-MT or thionein) exist as structurally flexible random coils. Depending on their environment, MTs may also exist in oxidized, polymerized, mixedmetal, and partially metalated forms.29 While apo-MTs are typically unstable toward oxidation and proteolytic degradation, the presence or addition of those metals that are natively bound was shown to stabilize MTs.30 Although it is now thought that most MTs have evolved to deal with Zn2+, Cd2+, and Cu+,26 in vitro studies with isolated mammalian MTs have shown their ability to bind metals as diverse as silver, gold, arsenic, bismuth, iron, cobalt, nickel, mercury, platinum, rhenium, and radioactive polonium and technetium.31−33 Moreover, the (over)expression of MTs is known to enhance tolerance against toxic metal ions including mercury, arsenic, platinum, lead, and cadmium in eukaryotes34 and bacteria.35−39 Importantly, several studies have indicated that MTs may also protect against uranium toxicity.7 In human kidney cells, exogenous MT inhibited DU-induced cell apoptosis.40 In the nematode Caenorhabditis elegans, MT synthesis is triggered by uranium, protecting against uranium toxicity and promoting uranium bioaccumulation.41 However, no molecular details on the protein level are available for any MT-uranium interactions; it is therefore of general interest to investigate whether and how an MT may directly interact with uranium. Cyanobacteria are a particularly attractive group of bacteria for metal capture from aqueous media.42−44 These photoautotrophs require only light, CO2, nitrogen, phosphate, and essential metal ions to thrive, and they are responsible for a substantial part of global CO2 fixation.45 Interactions of cyanobacteria with many metal ions have been studied,46−49 and it is thought that marine cyanobacteria play important roles for regulating the bioavailability of essential trace metal ions in seawater.50 Comparatively little is known about interactions between cyanobacteria and uranium; nevertheless, considering their abundance and global distribution, it is conceivable that they may also have importance for environmental uranium speciation. Hence, studies into the interactions of cyanobacteria with uranium are motivated by both fundamental questions and biotechnological applications.51−55 We have demonstrated bioremediation and bioextraction of uranium using cyanobacteria.53−55 Like other bacteria,12,13 cyanobacteria are able to capture uranyl on their cell surfaces. This biosorption involves negatively charged groups within the extracellular polysaccharides (EPS), or surface-associated polyphosphates.53,54,56 Like for bacteria in general, intracellular uranium metabolism has only recently begun to be explored.57 Recent work has shown that significant quantities (11.4 ± 0.1 mg/g dry cell weight; see Supporting Information for details) of uranium are taken up into the cytosol of Anabaena PCC sp. 7120 (C. Acharya et al., manuscript in preparation), opening questions regarding the intracellular fate, mode of action, and detoxification of this toxicant. One special feature of many cyanobacteria, including marine strains,46,58 is the presence of one or more MT gene in their genomes.59,60 In the freshwater strain Synechococcus sp. PCC7942, the smt operon codes for a 55-amino-acid MT (SmtA) and a zinc-responsive repressor
Figure 1. Solution NMR structure of the bacterial metallothionein Zn4SmtA from Synechococcus sp. PCC7942 (pdb 1jjd).62 Zinc ions are shown as purple spheres, sulfurs of the nine coordinating cysteines are shown as yellow spheres, and the two coordinating histidine nitrogens are shown as blue spheres. The secondary structure elements of the zinc finger fold are shown in red/gray (α-helix) and cyan (β-sheets). The “zinc finger” zinc site (A) is labeled with the numbers of the coordinating cysteines next to the respective sulfur atoms.
Heterologous overexpression of SmtA in E. coli conferred significant tolerance against uranium toxicity (Supporting Information, Figure S1). This observation has led us to explore possible in vitro interactions of uranyl with recombinantly expressed SmtA. The composition of complexes resulting from reactions with fully and partially metalated SmtA, as well as with completely demetalated apo-SmtA, was assessed using native electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). The structural consequences for SmtA following binding of UO22+ were analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopies. We found that SmtA, in its folded, metalated state, exhibited surprisingly high affinity toward the hard uranyl cation, likely using two bidentate surface sites composed of carboxylates. Attempts to observe reaction products of uranyl with demetalated SmtA led to complete oxidation and dimerization of the protein, with no metalated adducts detected. Similarly, the presence of substoichiometric quantities of Zn2+ during refolding yielded novel, partially metalated, and oxidized ZnMT species, but with little or no capacity for uranyl binding detected. Although the uranyl-binding surface sites we identified are not thought to have direct relevance to the normal biological function of SmtA, our findings may advance the understanding of uranyl−protein interactions in general, as surface-exposed sites similar to those in SmtA are likely to be widespread.
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RESULTS AND DISCUSSION Uranyl Binding to Recombinant Zn4SmtA. The in vitro reaction of recombinant Zn4SmtA with UO22+ was investigated by titrating Zn4SmtA (18 μM) with 0−50 mol equiv of UO22+ in the presence of 10 mM NH4HCO3 buffer at pH 7.8, monitored by ESI-MS (Figure 2A). The addition of increasing B
DOI: 10.1021/acs.inorgchem.5b02327 Inorg. Chem. XXXX, XXX, XXX−XXX
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where the reaction of a metal complex with an MT was studied by ESI-MS, namely, the reaction of cis- and trans-platin complexes with human Zn7MT-2, either retention or loss of Ndonor ligands was observable by ESI-MS.63 In that case, the coordination sphere of Pt(II) observed in the gas phase is likely to directly correspond to that in solution, as Pt(II)−N bonds are thermodynamically very stable and kinetically quite inert. In addition, the protonation of these donors (e.g., NH3) does not generate a neutral species that might be easily lost in the gas phase. In contrast, ligand exchange rates for uranyl are fast, and both hydroxo- and carbonato-ligands generate neutral, highly volatile species when protonated (H2O and CO2). It is therefore suggested that the small-molecule equatorial ligands become protonated during the electrospray ionization process and that they are lost together with bulk water and carbonate. In all cases, Zn4SmtA remained the most prominent species under the experimental conditions. Moreover, at no point was the loss of Zn2+ observed, indicating that UO22+ did not compete for Zn2+ binding sites. Furthermore, the ESI-MS data provide no evidence for any redox reaction involving the oxidation of thiols, which would lead to the loss of zinc and hydrogen.64 The acidification of these (UO2)nZn4SmtA preparations to pH 2.0 resulted in complete demetalation of the protein, with the major apo-SmtA mass peak at 5609.4 Da (data not shown). Since the titration involved relatively high molar ratios of UO22+, and also because ESI-MS is a gas-phase technique that may enhance ionic interactions, it was important to test how persistent the observed heterometallic species were in solution. Therefore, the incubations with 10 and 50 mol equiv of UO22+ were passed through a desalting gel filtration column equilibrated with 10 mM NH4HCO3, pH 7.8, followed by ultrafiltration. These preparations were subjected to ESI-MS and ICP-OES analyses. The deconvoluted ESI-MS mass spectrum of uranium−MT complexes resulting from addition of 10 mol equiv of UO2 2+ to Zn4SmtA (Figure 2B) showed a strong signal of Zn4SmtA (major species; 5863.7 Da; see Table 1 for theoretical masses) but had retained a clear minor signal for (UO2)Zn4SmtA (6131.8 Da). The spectrum for the incubation with 50 mol equiv also showed this species along with additional small peaks for (UO2)2Zn4SmtA (6400.4 Da; theoretical: 6399.76 Da) and
Figure 2. Deconvoluted native ESI-MS spectra of (UO2)nZn4SmtA complexes formed in the presence of increasing molar ratios (5, 10, 20, 50) of UO2/Zn4SmtA before (A) and after (B) gel filtration and ultrafiltration (18 μM protein, 10 mM NH4HCO3, 10% MeOH, pH 7.8).
amounts of UO22+ led to the formation of heterometallic species of the form (UO2)nZn4SmtA, where n = 1−3. Each new neutral mass peak differed by 268 Da from the preceding peak, consistent with the addition of 238UO22+ and simultaneous loss of two H+ to maintain formal charge neutrality. No adducts incorporating any small-molecule equatorial ligands were observed. In one of the very few other reported studies
Table 1. Average Neutral Massesa of Species Observed after Reaction of apo-SmtA with Different Ratios of Zn2+ and UO22+ observed neutral average mass (Da) species apo-SmtA Zn1SmtA Zn2SmtA Zn3SmtA Zn4SmtA (UO2) Zn3SmtA (UO2) Zn4SmtA fully oxidized dimer
theoretical neutral average mass (Da) 5610.24 5673.61 5736.99 5800.36 5863.77 6068.37 6131.75
(4Zn)
(3Zn/1U) 5670.40 (−3.21) 5735.19 (−1.80) 5800.02 (−0.34)
(2Zn/2U) 5606.30 5670.36 5735.07 5800.00
(−3.94) (−3.25) (−1.86) (−0.36)
(1Zn/3U)
(4U)
(no metal)
5601.27 (−8.97)
5601.0 (−9.24)
11203.00 (−0.34)
11201.8 (−0.54)
5670.45 (−3.16) 5735.14 (−1.86) 5799.32 (−1.04)
5863.60 (−0.17) 6068.10 (−0.27) 6131.16 (−0.59)
11202.34
a
Theoretical average masses were calculated using ISOTOPIDENT (http://education.expasy.org/student_projects/isotopident/htdocs/), assuming fully reduced thiols unless stated otherwise. Numbers in brackets indicate the deviation from the theoretical mass; for undermetalated species, deviations of ∼2 Da and above indicate disulfide bond formation, which is associated with the loss of hydrogen. C
DOI: 10.1021/acs.inorgchem.5b02327 Inorg. Chem. XXXX, XXX, XXX−XXX
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presence of reduced thiols from glutathione and a range of redox-active proteins including MTs. On the basis of typical redox potentials of cells and that of the U(IV)/U(VI) couple at neutral pH (0.05 to −0.2 V; the latter value is valid in the presence of carbonate),68 it can be concluded that uranyl will become reduced to U(IV) once inside a cell. Indeed, reduction to U(IV), with concomitant changes in the ratio of reduced and oxidized glutathione, has been demonstrated for Brassica napus cells.69 Hence, organic thiols, including those present in MTs, may in principle reduce uranyl to U(IV), which could lead to different interactions with an MT. The redox potentials of MTs are heavily dependent on whether or not they are metal-bound, with demetalation rendering them much more reducing than in their metalated state (e.g., redox potential vs Ag/AgCl −0.6 V for apo-MTs, and −0.38 V for Zn-MTs).70 It has become widely accepted that in vivo, MTs do not necessarily occur in their fully metalated forms, and antioxidant properties of some MTs are well-documented.25 On the basis of these considerations, it was of interest to explore the interactions of completely and partially demetalated SmtA with UO22+. Reactions of apo-SmtA with UO22+. Apo-SmtA was generated from Zn4SmtA by reducing the pH to 1−2 with subsequent separation of protein and metal ions by rapid gel filtration chromatography under nitrogen. Demetalated apoSmtA was reacted at pH 2 with 4 mol equiv of either Zn2+ or UO22+, and the species generated after reneutralization (to pH 7.8) were analyzed by ESI-MS (Figure 4).
(UO2)3Zn4SmtA (6668.3 Da; theoretical: 6667.77 Da) (Figure 2B). Analysis of protein and metal contents for the latter two preparations by ICP-OES gave a value of 1.1:1.0 UO2/SmtA for 10 mol equiv of uranyl, and 1.75:1.0 for 50 mol equiv of uranyl, in both cases significantly higher than suggested by ESI-MS. These findings from two independent methods indicate that the interaction of UO22+ with SmtA is sufficiently strong to withstand gel filtration and ultrafiltration, even in the presence of a very large (over 500-fold) excess of (hydrogen)carbonate. To appreciate the significance of this observation, it is important to consider that uranyl forms rather strong complexes with carbonate. Speciation modeling using published stability constants82 indicates that in the absence of protein, at pH 7.8 and 10 mM (hydrogen)carbonate, 100% of 1 mM uranyl is engaged in carbonate complexes, predominantly the [UO2(CO3)3]4− complex (Supporting Information, Figure S2). Figure 3 shows competition experiments between Zn4SmtA
Figure 3. Competitive binding of uranyl to Zn4SmtA in the presence of varying concentrations of carbonate (20 μM Zn4SmtA, 20 μM UO22+, 10 mM Tris-Cl, pH 8.1). Data were fitted using a Hill-type function; the midpoint is at 0.23 mM carbonate. Combining the data reported in this Figure with speciation modeling (Supplementary Figure S2) gives a KD for uranyl binding by SmtA of 100 pM.
and (hydrogen)carbonate. These data allow the determination of a conditional affinity constant of log K = 10.0 ± 0.6 at pH 8.1, (i.e., KD = 100 pM). It is also noteworthy that the formation of the (UO2)nZn4SmtA species was rapid: incubation with uranyl (ranging from 18 μM to 1 mM) for either 5−10 min or overnight yielded similar results. This is in line with expectations, as ligand-exchange reactions with uranyl are known to be relatively fast.65 In summary, although UO22+ was not able todirectly or indirectly through thiol oxidationdisplace Zn2+ from SmtA, heterometallic complexes of significant stability were formed, which competed effectively with a large excess of (hydrogen)carbonate. The fact that UO22+ did not compete for zinc binding sites is not surprising, given that UO22+ is a hard Lewis acid with very little affinity to thiolates.65 In contrast, softer U(IV) has been shown to form homoleptic tetra- and hexathiolate complexes, in the absence of oxygen.66,67 The cytosol of cells is a generally reducing environment due to the exclusion of oxygen and the
Figure 4. Products of the reaction of apo-SmtA with either Zn2+ or UO22+ (18 μM SmtA, 10 mM NH4HCO3, 10% MeOH). To illustrate dimer formation, raw (nondeconvoluted) ESI-MS spectra are shown, with charge states indicated. (A) Reconstitution of apo-SmtA with 4 mol equiv of Zn2+ leads to the observation of peaks for monomeric Zn4SmtA only (pH 7.8). (B) Acidification of this preparation to pH 2.0 generates monomeric, reduced apo-SmtA. (C) Neutralization of apo-SmtA in the presence of 4 mol equiv of UO22+ generates exclusively metal-free species (pH 7.8). The abundant species at 1601.26 m/z (shaded box) corresponds to the apo-dimer, which is formed in the absence of Zn2+. Observed deconvoluted and theoretical masses are compiled in Table 1. D
DOI: 10.1021/acs.inorgchem.5b02327 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry In the control reaction, apo-SmtA reconstituted with 4 mol equiv of Zn reproduced the native Zn4SmtA species with a mass of 5863.6 Da for the neutral species (Figure 4A). In agreement with this, the molar ratio of Zn to SmtA calculated from zinc and sulfur contents measured by ICP-OES was found to be 3.8:1. On being acidified with formic acid to pH 2, reconstituted Zn4SmtA was demetalated and showed a mass of 5609.4 Da consistent with fully reduced apo-SmtA (Figure 4B; see Table 1 for theoretical masses). Apo-SmtA incubated with 4 equiv of UO22+ resulted in the formation of an oxidized apo-SmtA dimer with a neutral mass of 11202.2 Da (Figure 4C; Table 1), with all nine cysteine thiols engaged in intra- and (because of the uneven number and suggested by the observation of a dimer) at least one intermolecular disulfide bond. The relative intensities of the various charge states suggest that a mixture of (oxidized) monomer and dimer was obtained. The concentration of free reduced thiols estimated by Ellman’s method using 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB) for this preparation was found to be negligible confirming complete oxidation of thiols. Neither uranium nor zinc was found to be associated with this form of SmtA as confirmed by ICP-OES analyses. A control experiment with no added metals, in the presence of air, also led to the formation of an oxidized apo-dimer (Supporting Information, Figure S3). The ESI-MS studies were supported by optical spectroscopy and SDS-PAGE analyses of reconstituted metalloprotein solutions. The far-UV CD spectrum of native Zn4SmtA (Figure 5A) revealed minima at 208 and 228 nm and a maximum at 193 nm, consistent with the presence of a short α helix, several short β strands, and extensive random-coil stretches, as observed for the solution NMR structure of Zn4SmtA (Figure 1).62 While the reconstituted Zn4SmtA exhibited a CD fingerprint very similar to that of native Zn4SmtA, the apo-SmtA monomer/dimer preparation (resulting from the reaction of apo-SmtA with 4 mol equiv of UO22+) displayed a spectrum corresponding to all-random coil conformation, with a minimum at ∼200 nm (Figure 5A). This is in line with expectations, as MTs require metal ions for folding, and accordingly, other apo-MTs have been reported to exist in random coil conformation.26,28,32 The reconstituted Zn4SmtA, diluted in Laemmli sample buffer containing β-mercaptoethanol and SDS, and heated at 95 °C for 5 min, demonstrated a profile similar to native SmtA on SDS-PAGE (Figure 5B), with two bands at ca. 70 and 100 kDa observed. While these are well above the expected mass, the erratic migration behavior of MTs is well-known,71 and it can be attributed to incomplete denaturation. In contrast, the oxidized apo-SmtA sample treated in the same way revealed a band close to the molecular weight expected for monomeric SmtA (Figure 5B). This indicates that at least the intermolecular disulfide bonds may be easily reduced; the latter conclusion is also supported by the finding that incubations of oxidized apo-SmtA with a 40-fold molar excess of DTT at 4 °C overnight resulted in the complete disappearance of the dimer as analyzed by ESI-MS (Supporting Information, Figure S4). To shed some light as to the likely site(s) of dimerization, samples containing dimeric apo-SmtA were digested with trypsin and analyzed by ESI-MS (Figure 6). Clean spectra were obtained, with monoisotopic mass peaks at m/z 474.3 (A23IDR26), 749.5 (T2STTLVK8), 965.9 (N27− K45, with two oxidized Cys; [M+2H+]2+), 985.4 (dimeric C9−
Figure 5. CD and SDS-PAGE analyses of apo-SmtA after reaction with metals. (A) CD spectra recorded for reconstituted Zn4SmtA (-------) and apo-SmtA after reaction with 4 mol equiv of uranyl (•••••). A control spectrum for native Zn4SmtA as isolated from E. coli () is also included. (B) SDS-PAGE analyses of SmtA. Lane 1: molecular weight marker, lane 2: native Zn4SmtA, lane 3: Apo-SmtA after reaction with 4 equiv of Zn2+, lane 4: Apo-SmtA after reaction with 4 equiv of UO22+, lane 5: Apo-SmtA after reaction with 4 equiv of UO22+ followed by boiling in the presence of β-mercaptoethanol for 1 h (all four samples were heated for 5 min in Laemmli sample buffer; see text for further details).
K22, with eight oxidized Cys; [M+3H+]3+), 1042.3 (dimeric G46−G56, with six oxidized Cys; [M+2H+]2+), 1477.6 (C9−K22, with four oxidized Cys; [M+H+]+, and also C9−K22, with eight oxidized Cys; [M+2H+]2+). Accordingly, intermolecular disulfide bonds were found for two different fragments, suggesting that there are at least two different dimerization sites. To summarize, the results from reacting apo-SmtA with UO22+ indicate that unfolded, completely zinc-free apo-SmtA has no inherent binding capacity for UO22+ or any potentially formed U(IV). Reactions of Partially Metalated SmtA with UO22+. SmtA is an unusual MT, as in its Zn4 form, its first 38 residues adopt a defined zinc finger fold, held together by zinc(A), which is bound to Cys9, 14, 32, and 36 (Figure 1). This zinc finger scaffold is likely essential for providing a binding pocket for the other three zinc ions. To investigate whether undermetalated SmtA may provide binding sites for either UO22+ or potentially reduced U(IV), purified apo-SmtA was also reacted with mixtures of UO22+ and Zn2+ ions in various stoichiometric ratios. As before, excess metal ions were separated from these metalated protein complexes by gel filtration and ultracentrifugation before analysis by mass spectrometry (Figure 7). In all three preparations (molar ratios Zn/U = 3:1, 2:2, and 1:3), undermetalated zinc complexes (Zn 1 , Zn2 , Zn 3 ) dominated the speciation, with their relative abundances varying in dependence on added Zn2+. For instance, at the E
DOI: 10.1021/acs.inorgchem.5b02327 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 7. Products of reactions of apo-SmtA with different ratios of Zn2+ and UO22+, as analyzed by native ESI-MS (18 μM protein, 10 mM NH4HCO3, 10% MeOH, pH 7.8). Zn/UO2 ratios are indicated in the Figure. Observed masses are reported in Table 1, together with all theoretical masses.
content, the higher is the degree of oxidation; this suggests that at least some of the thiols that are not engaged in zinc binding are oxidized. Inspection of the raw MS data (not shown) indicated that the majority of the species was in monomeric form. In summary, the only species that offered appreciable binding capacity for UO22+ were the Zn4SmtA and Zn3SmtA species (Figures 2 and 7). This suggests that SmtA needs to be in an essentially well-folded state to provide at least one uranyl binding site. We therefore next explored the effect of UO22+ on the structure of Zn4SmtA and the most likely site(s) for uranylSmtA interactions. Structural Effects of Uranyl Binding and Likely Binding Sites. The impact of UO22+ additions on the secondary structure of Zn4SmtA was analyzed by CD spectroscopy at pH 7.4. Stepwise additions of UO22+ (1−20 mol equiv ) had no major effect on the CD spectrum (Figure 8A); the very small reduction in the intensity of the minimum at 208 nm is likely due to small variations in concentration. The effects of uranyl on protein folding and on individual amino acid residues were investigated using one-dimensional (1D) and two-dimensional (2D) 1H NMR spectroscopy. After addition of a two-fold excess of UO22+ to Zn4SmtA in the absence of carbonate, several 1D spectra were recorded over a period of 5 h, to ensure that equilibrium had been reached, before recording a 2D TOCSY spectrum. The 1D 1H spectrum of Zn4SmtA was largely unaffected, but three resonances between 2.1 and 2.6 ppm were selectively broadened immediately after mixing, and they remained broadened over the entire course of the NMR experiments (15 h) (Figure 8B). As a complete sequential assignment for Zn4SmtA is available,62 it was possible to identify the γ-protons of Glu12 and Glu34 as well as the β-protons of Asp38 as the affected nuclei. This is consistent with an interaction of the hard UO22+ moiety with the carboxylate groups of these residues. Inspection of the electrostatic surface of Zn4SmtA (Figure 8C) suggested that Glu34 and Asp38 may provide a bidentate binding site for UO22+ and that a second bidentate site may be formed by
Figure 6. ESI-MS spectrum of a tryptic digest of dimeric apo-SmtA. All masses (M) given are neutral monoisotopic masses and agree within
