Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine

Abstract The ability to specifically label the sulphide ions of protein-bound iron–sulphur (FeS) clusters with 34S isotope greatly facilitates structure–function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the O-acetyl-l-serine sulfhydrylase CysK to generate 34S-substituted l-cysteine and subsequently use it as a substrate for the l-cysteine desulfurase NifS to gradually supply 34S2− for in vitro FeS cluster assembly in an otherwise standard cluster reconstitution protocol.


Introduction
Proteins that contain iron-sulphur (FeS) clusters are extremely widespread in nature and play key roles in an array of biochemical processes from respiration and photosynthesis to DNA replication. They contain iron and inorganic sulphide in structural arrangements that differ in nuclearity as well as shape, for example, the rhombic [2Fe-2S] and cubic [4Fe-4S] clusters [1]. Cysteine thiolates (RS À ) are by far the most common amino acid ligands to FeS clusters, but other residues such as histidine (-N¼), serine (R-O À ) and aspartate (R-CO 2 À ) are known [2]. In addition to roles in electron transfer, redox and Lewis acid catalysis, the inherent reactivity of FeS clusters with a range of small molecules makes them ideal candidates for sensing environmental changes and stresses caused by reactive oxygen (ROS) and/or nitrogen (RNS) species. FeS cluster containing transcriptional regulators have evolved to exploit this sensitivity as a way of modulating protein-DNA interactions and hence a means to effect transcriptional regulation [3]. Recent advances in the purification and handling of extremely sensitive FeS proteins at high concentration have facilitated the application of a range of biophysical techniques to study the nature and reactions of the FeS cluster with ROS/RNS. The use of stable isotopes, particularly of iron ( 57 Fe), sulphur ( 34 S), and nitrogen ( 15 N in NO) have proved instrumental in these advancements, for example, via mass spectrometry and nuclear resonance vibrational spectroscopy (NRVS) [4][5][6][7][8].
We previously described a method for the in vivo incorporation of 57 Fe into FeS clusters [9]. However, it is not cost-effective to attempt large-scale 34 S-labeling in vivo. Fortuitously, in many cases, FeS clusters will self-assemble, in vitro. Addition of ferrous and sulphide salts is one approach, which works well in some cases but can be difficult to control, leading to black FeS precipitates or adventitious, non-physiological protein-associated FeS species. A more refined biochemical method that is widely used by researchers in the FeS protein field employs an enzyme naturally involved in FeS cluster assembly in vivo [10]. The simplest of these utilizes a cysteine desulfurase, typically Azotobacter vinelandii NifS, as a way of gradually generating sulphide from L-cysteine [11][12][13]. NifS reconstitutions typically result in a good recovery of holo-protein (!70%) with spectroscopic features indistinguishable from those of the in vivo-derived counterpart [10,14].
Currently, 34 S-L-cysteine is not commercially available. Two in vitro methods for the total synthesis of L-cysteine have been reported. The first, in which elemental sulphur is reacted with benzyl-magnesium chloride and L-b-chloroalanine prior to the formation of cysteine, provides poor yields of a crucial intermediate in the process, S-benzylcysteine [15,16]. The second, involving the reaction of thioacetic acid with a-acetamidoacryl acid, results in a racemic mixture of D-and L-cysteine [17]. In vivo, L-cysteine is synthesized from L-serine by the action of the two enzymes CysE and CysK [18]. CysE (EC 2.3.1.30) is a L-serine O-acetyltransferase, while CysK (EC 2.5.1.47) is a pyridoxal-5 0phosphate dependent enzyme with O-acetyl-L-serine (OAS) sulfhydrylase activity, catalysing the stereo specific formation of L-cysteine via a nucleophilic addition of inorganic sulphide to OAS [19]. We note that naturally occurring D-cysteine is synthesized by a dedicated L-to D-amino acid racemase (EC 5.1.1.10) distinct from CysK.
Here, we build on the method of Ono et al. [20] for synthesizing 34 S-L-cysteine from commercially available OAS by employing a thermostable CysK, from Geobacillus stearothermophilus [21], and demonstrate its utilization in the preparation of 34 S-labeled FeS clusters in a number of FeS clusters containing transcriptional regulators.

Preparation of CysK
Luria-Bertani medium (2 Â 500 ml) was inoculated with freshly transformed BL21 kDE3 Escherichia coli containing the CysK expression vector [pET11a encoding a codon optimized cysK gene (uniprot id: Q84IF9) from the thermophile G. stearothermophilus cloned using NdeI and BamHI sites; Genscript]. Geobacillus stearothermophilus CysK was selected because it has been previously characterized [21], and its thermal stability and general robustness provides enhanced flexibility for possible future applications. Ampicillin (100 mg/ml) was added and the culture grown at 37 C, 200 rpm, until OD 600 nm reached $0.8. Expression was induced by the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG, 1 mM). After 20 min, the cultures were supplemented with 50 mM L-methionine, 50 mM glycine, 50 mM (NH 4 ) 2 SO 4 and Minimum Essential Medium (MEM) vitamins (10 ml/ml, containing pyridoxal hydrochloride; a precursor to pyridoxal-5 0 -phosphate, 100Â, Sigma Aldrich) and incubated for a further 4 h at 37 C. The cells were harvested by centrifugation at 10 000Âg for 15 min at 4 C. Cell pellets were resuspended in 40 ml of buffer A (50 mM Tris HCl, pH 7.0) and sonicated twice while on ice, and then centrifuged at 40 000Âg for 45 min at 1 C. The cleared cell lysate was treated with streptomycin sulphate (15 mg/ml), incubated on ice for 30 min, and then centrifuged at 20 000Âg for 15 min at 1 C. The supernatant was then fractionally precipitated with ammonium sulphate between 50% and 80% saturation [22], before centrifugation at 20 000Âg, as described above. The precipitate from the 80% saturation step was dissolved in 15 ml of buffer A and dialysed (10 kDa MWCO) overnight at 4 C against 1 L buffer A.
Post dialysis, the sample was loaded onto a Q Sepharose column (15 ml) and washed with buffer A containing 3% (v/v) buffer B (50 mM Tris HCl 800 mM NaCl, pH 7.0). Bound proteins were eluted using a 50 ml linear gradient between 3% and 32% (v/v) buffer B. Fractions (1 ml) containing CysK were pooled, incubated at 65 C for 30 min, centrifuged at 17 000Âg for 15 min at room temperature, diluted 5-fold with buffer A and concentrated, as previously described [9], using a Q Sepharose column (1 ml). CysK was eluted using buffer B, separated into 50 ml aliquots and stored at À80 C until needed. Purity of the final preparation was $95%, as judged by SDS-PAGE. Protein concentration was determined by the method of Bradford (BioRad) [23], with bovine serum albumin as the standard.

Preparation of NifS
Azotobacter vinelandii NifS was purified largely as previously described [11]. Briefly, E. coli BL21(DE3) cells containing the nifS expression plasmid pDB551 were grown at 37 C, 200 rpm in Luria broth containing ampicillin (100 mg/l). NifS production was induced when cells reached A 600 nm ¼ 0.6 by the addition of 1 mM IPTG. Cultures were supplemented with 1Â MEM vitamins 10 min post induction and further incubated for 2 h. Following harvesting, cell pellets were resuspended in Tris buffer (25 mM Tris pH 7.4), lysed by sonication and centrifuged. Solid streptomycin sulphate was added to the supernatant (1.5 g/100 ml), which was incubated on ice for 15 min. The resulting suspension was centrifuged and the supernatant fractionated using ammonium sulphate, with NifS precipitating in the 25-45% cut. NifS was dissolved in Tris buffer, dialysed (10 kDa MWCO) overnight at 4 C against 1 l of Tris buffer. Post diaslysis, the sample was loaded on to a 10 ml HiTrapQ Sepharose column (GE Healthcare) and eluted using a 0.1-0.6 M NaCl gradient, yielding NifS at $95% purity, as judged by SDS-PAGE. The sample was aliquoted (20 ml) and stored at À80 C until needed. Protein concentration was determined as above.
Reduction of S 0 to sulphide 34 S-sulphur (98% enrichment, Cambridge Isotope Laboratories, Goss Scientific) was reduced to sulphide (S 2À ) by the action of sodium metal in liquid ammonia via a Schlenk line as previously described [24,25]. Briefly, an aliquot ($135 mg) of sodium metal was dissolved in liquid ammonia. To this was added an aliquot ($100 mg) of 34 S-sulphur, maintaining a molar stoichiometry of approximately two sodium per sulphur. During the reaction the blue colour typical of solvated electrons faded, after which the liquid ammonia was allowed to evaporate under a stream of nitrogen. The remaining residue was carefully dissolved in a minimal volume of anaerobic 25 mM NaOH and passed through a 0.2 mm filter to remove any particulates. The resulting sulphide solution was assayed according to the method of Beinert [26].

FeS cluster reconstitution
FeS proteins (E. coli FNR, Streptomyces coelicolor NsrR and Rhizobium leguminosarum RirA) were purified as previously described [4][5][6][7]. Naturally incorporated FeS clusters were removed by dialysis in the presence of air. Reconstitution of the FeS cluster was carried out with NifS, as previously described [9], except that 34 S-L-cysteine solution was used in place of natural abundance L-cysteine. Briefly, anaerobic apo-protein (in the range 70-500 mM) was treated with a 16-fold excess of 34 S-L-cysteine, 40-fold excess of dithiothreitol, and up to a 10-fold excess of (NH 4 ) 2 Fe(SO 4 ) 2 over the apo-protein. The buffer used was dependent on the protein: FNR, 25 mM HEPES, 2.5 mM CaCl 2 , 100 mM NaCl, 100 mM NaNO 3 , pH 7.5; NsrR, 50 mM Tris, 50 mM NaCl, 5% (v/v) glycerol, pH 8.0; RirA, 25 mM HEPES, 2.5 mM CaCl 2 , 50 mM NaCl, 750 mM KCl, pH 7.5. Azotobacter vinelandii NifS ($225 nM) was added and the reaction mixture incubated with stirring at 20-37 C, depending on the protein (FNR, 37 C; NsrR, 30 C; RirA, 20 C). UV-visible absorbance spectra were recorded every 20 min until no further increases in absorbance due to the FeS cluster were apparent (the time required varies between proteins but is typically complete after a few hours). Low molecular mass contaminants were removed by applying the reconstitution reaction mixture to a 1 ml HiTrap heparin column (GE Healthcare) and eluting with a NaCl gradient of 100-500 mM in the same buffer as above. For non-DNA-binding FeS proteins, a Sephadex G25 column (PD10, GE Healthcare) can be used instead.
Electrospray ionization mass spectrometry (ESI-MS) of proteins under non-denaturing conditions in ammonium acetate buffer was performed using a Bruker microOTOF-QIII mass spectrometer operating in the positive ion mode and calibrated using ESI-L Low concentration tuning mix, as previously described [4][5][6]. Processing and data analysis were carried out using Compass Data Analysis version 4.1. Neutral mass spectra were generated using ESI compass Maximum Entropy deconvolution algorithm version 1.3. Exact masses are reported from peak centroids representing the isotope average neutral mass. For apo-proteins, these are derived from the m/z spectra, for which peaks correspond to [M þ zH]/z. For cluster-containing proteins, where the cluster contributes charge, the peaks correspond to [M þ (FeS) nþ þ (zÀn)H]/z, where M is the molecular mass of the protein, FeS is the mass of the FeS cluster of nþ charge, H is the mass of the proton and z is the total charge. In the expression, the nþ charge of the FeS cluster offsets the number of proteins required to achieve an observable charge state with z charges [32,33]. Predicted masses are given as the isotope average of the neutral protein in which FeS cluster binding is expected to be charge compensated.

Synthesis of 34 S-L-cysteine by CysK
The ability to specifically isotopically label the acid labile sulphides of FeS clusters is a powerful tool for enhancing studies using spectroscopic techniques, such as resonance Raman spectroscopy and nuclear vibrational resonance spectroscopy. These methods probe vibrations involving sulphur species where the increase in mass due to incorporation of 34 S results in a decrease in the energy of vibrational bands, enabling the deconvolution of cluster Fe-S and Fe-cysteinyl contributions to the vibrational spectrum [6,7]. It is also extremely useful for studies by non-denaturing mass spectrometry, in which assignments of FeS clusters and their conversion/degradation products can be made unambiguously through the detection of mass shifts [5].
The first step towards a simple method to achieve specific isotopic substitution of cluster sulphide was the synthesis of 34 S-L-cysteine. This was achieved according to Ono et al. [20], except CysK from G. stearothermophilus was used to catalyse the reaction between Na 34 2 S and O-acetyl-L-serine at 50 C overnight [6,7]. After removal of enzyme, the reaction mixture contained no detectable protein or sulphide, indicating complete reaction and this was confirmed by reaction with DTNB, which produced the characteristic yellow colour indicative of the presence of thiolate species. TLC plates developed with DTNB yielded a single spot with a R f value of 0.25 (6 0.03), which is consistent with that of L-cysteine (0.29). Plates developed with an amine-specific reagent, ninhydrin, revealed two spots with R f values of 0.25 (6 0.03) and 0.51 (6 0.03), corresponding to cysteine and unreacted OAS, respectively. LC-MS of purified 34 S-L-cysteine revealed a single major [M þ H] 1þ peak at m/z ¼ 124.019, corresponding to a mass of 123.011 g/mol, very close to the expected monoisotopic mass of 34 S-cysteine 123.016 g/mol ([M þ H] 1þ m/ z ¼ 124.023) and shifted by approximately þ2 g/mol relative to that of natural abundance cysteine (Fig. 1). Addition of the reaction mixture to a sample of NifS caused the major absorbance peak at 392 nm to shift to 416 nm with the concomitant appearance of a band at 370 nm (not shown), consistent with the presence of cysteine, as previously described [11]. O-acetylserine alone did not alter the spectrum of NifS and we note that Dcysteine is not a substrate for the enzyme [11].

Reconstitution of FeS clusters containing 34 S-sulphide
The reconstitution of FeS clusters using the cysteine desulfurase NifS is well known to be more efficient than equivalent reactions employing a sulphide salt as the source of sulphur, presumably due to the gradual production of sulphide that minimizes formation of unproductive iron-sulphide precipitates [11][12][13]. Here, using a standard procedure with 34 S-L cysteine in place of natural abundance cysteine, apo-protein forms of E. coli FNR [7], S. coelicolor NsrR [30], and R. leguminosarum RirA [4] were reconstituted to generate FeS cluster (holo) proteins. Resulting samples were typically !70% cluster loaded [5][6][7].
It is important to note that FeS proteins readily form sulphur adducts, usually involving the insertion of cluster-derived sulphide that has undergone oxidation to form sulphane (S 0 ), which in turn can incorporate into the thiolate side chain of cysteine residues to form persulphides adducts [5,7,34]. The preparation of clean, persulphide-free apo-protein is an important prerequisite for preparation of 34 S-labelled FeS proteins (particularly when mass spectrometry studies are planned) and so Tris (2-carboxyethyl) phosphine (TCEP) was used here prior to reconstitution [35]. Post reconstitution, proteins were separated from low molecular weight species via a combination of weak ion exchange and/or gel filtration techniques; in some cases, this can selectively enrich the holo-protein content of the sample [9]. It is important to compare the biophysical properties of the in vitro reconstituted protein to those of in vivo assembled material, wherever possible [10,14]. CD spectroscopy is ideally suited for this purpose, as the electronic transitions that underlie the broad absorption spectrum of many FeS proteins can be resolved via CD spectroscopy. This optical activity arises from the asymmetric protein fold to which the FeS cluster is ligated. The CD spectrum can be used to ensure the quality of the FeS protein samples between preparations, that is, for natural abundance and 34 S-labelled preparations of the same protein. Figure 2 shows the anaerobic CD spectra of [4Fe-4S] FNR assembled in vivo and reconstituted in vitro with and without 34 S-Lcysteine in place of regular cysteine. The CD spectra all display three major positive features at 330, 380 and 420 nm with comparable De values indicating the [4Fe-4S] 2þ clusters in each preparation are in essentially identical environments [9].

Mass spectrometric determination of 34 S incorporation into FeS clusters
During ESI-MS biological analytes are introduced into the mass spectrometer in a non-denaturing volatile aqueous solvent, giving rise to multiply charged ions in the gas phase that preserve the non-covalent interactions found in protein-protein and protein-cofactor complexes [36][37][38]. This non-denaturing ESI-MS technique is finding increasing application in the characterization of a wide range of metalloproteins [39,40], including a growing number of FeS proteins [4,5,32,41]. The strength of the technique lies with its ability to identify, as well as determine the stoichiometry of, protein-associated metal (and sulphide) ions. We have recently applied time-resolved ESI-MS to the study of FNR, a master regulator controlling the switch between anaerobic and aerobic respiration in E. coli and many other bacteria [5]. In 34 S is þ7.6 Da (taking into account the natural abundance of sulphur isotopes). To demonstrate further the general utility of the methodology, 34 S substituted forms of two other FeS containing transcriptional regulators, S. coelicolor NsrR and R. leguminosarum RirA, were generated ( Fig. 3b and c, respectively). In each case, full incorporation of 34 S was demonstrated through the observation of a þ8 Da mass shift compared to the mass observed for the  The buffer was 25 mM HEPES, 2.5 mM CaCl2, 100 mM NaCl, 100 mM NaNO3, pH 7.5. Note that the S24F variant of FNR was employed in time resolved ESI-MS studies of the FNR cluster conversion mechanism [5].
protein containing a cluster with natural abundance sulphur. For NsrR, the [4Fe-4S] peak shifted from 17 823 to 17 831 Da (Fig. 3b); for RirA, the shift was from 17 792 to 17 800 Da (Fig. 3c). Note; for clarity, the mass range shown in Fig. 3 has been restricted to the area immediately either side of the main [4Fe-4S] protein peak in the monomeric region of the spectrum to highlight the 34 S induced mass shift. Full mass spectra for FNR, NsrR, and RirA containing naturally abundant [4Fe-4S] clusters have been published elsewhere [4,5,30].

Conclusions
Here, we describe a convenient and generally applicable method for specifically labelling the sulphides of FeS cluster proteins with 34 S. The method, which is based on two enzymecatalysed reactions, avoids the problems associated with direct chemical reconstitution of FeS cluster proteins by providing regulated amounts of 34 S 2À for cluster assembly. The resulting 34 Slabeled FeS clusters greatly facilitate structural and mechanistic studies, as already demonstrated through resonance Raman [7], NRVS [6], and ESI-MS studies [5].