A [3Fe-4S] cluster is required for tRNA thiolation in archaea and eukaryotes

Significance

Posttranscriptional transfer RNA (tRNA) modifications with sulfur atoms have important roles in accurate and efficient translation and stress responses. Although the enzymes responsible for 2-thiouridine (s²U) and 4-thiouridine (s⁴U) modifications have been identified in archaea and eukaryotes, their catalytic mechanisms remain unclear because of the complexity of the sulfur transfer process. This study demonstrates that the sulfur insertion enzymes that catalyze the synthesis of s²U in archaea and eukaryotic cytosols, as well as s⁴U and Cys-tRNA in methanogenic archaea, all contain a [3Fe-4S] cluster. This finding provides important insights into sulfur transfer reaction mechanisms and establishes a direct link between the ancient Fe-S cluster to translation in archaea and eukaryotes.

Abstract

The sulfur-containing nucleosides in transfer RNA (tRNAs) are present in all three domains of life; they have critical functions for accurate and efficient translation, such as tRNA structure stabilization and proper codon recognition. The tRNA modification enzymes ThiI (in bacteria and archaea) and Ncs6 (in archaea and eukaryotic cytosols) catalyze the formation of 4-thiouridine (s⁴U) and 2-thiouridine (s²U), respectively. The ThiI homologs were proposed to transfer sulfur via cysteine persulfide enzyme adducts, whereas the reaction mechanism of Ncs6 remains unknown. Here we show that ThiI from the archaeon Methanococcus maripaludis contains a [3Fe-4S] cluster that is essential for its tRNA thiolation activity. Furthermore, the archaeal and eukaryotic Ncs6 homologs as well as phosphoseryl-tRNA (Sep-tRNA):Cys-tRNA synthase (SepCysS), which catalyzes the Sep-tRNA to Cys-tRNA conversion in methanogens, also possess a [3Fe-4S] cluster similar to the methanogenic archaeal ThiI. These results suggest that the diverse tRNA thiolation processes in archaea and eukaryotic cytosols share a common mechanism dependent on a [3Fe-4S] cluster for sulfur transfer.

Sulfur atoms are present in several modified transfer RNA (tRNA) nucleosides, such as 4-thiouridine (s⁴U), 5-methyl-2-thiouridine derivatives (xm⁵s²U), 2-thiocytidine (s²C), and 2-methylthioadenosine (ms²A) derivatives. The s⁴U nucleoside is found at position 8 of most bacterial and archaeal tRNAs and functions as a photosensor for near-UV irradiation (1). Upon irradiation, the s⁴U8 cross-links with the nearby cytidine at position 13. This reaction causes conformational changes and prevents aminoacylation of tRNAs, resulting in accumulations of uncharged tRNAs that trigger stringent responses (1). The xm⁵s²U nucleosides are present at position 34 (the wobble base) of tRNA^Gln_UUG, tRNA^Glu_UUC, and tRNA^Lys_UUU in all three domains of life. The rigid conformation of xm⁵s²U―preferentially in the C3′-endo form―ensures efficient codon:anticodon base pairing (1, 2). The presence of s²U in the codon:anticodon pair leading to a preference for A-ending codons (3) may be explained by the greater stability of the s²U-A vs. s²U-G pair (4). Furthermore, the 2-thio group of xm⁵s²U acts as an identity element in aminoacylation reactions (5–7), promotes tRNA binding to the ribosomal A-site (7), and prevents frameshifting during translation (8). Yeast mutants lacking the 2-thio modification have pleotropic phenotypes, such as defects in invasive growth (9), hypersensitivity to high temperature, rapamycin, caffeine, or oxidative stress (10, 11), inability to maintain normal metabolic cycles (12), and protein misfolding and aggregation (13). In humans, impaired 2-thio modification of the mitochondrial tRNAs has been associated with acute infantile liver failure (14, 15) and myoclonic epilepsy with ragged-red fibers (16, 17). Overall, s⁴U8 and s²U34 in tRNAs play important roles in translation.

The sulfur transfer processes for s⁴U8 and s²U34 biosynthesis are complicated, and some of the details (especially in archaea and eukaryotes) remain unclear because: (i) they usually involve a cascade of sulfur carrier proteins rather than a direct transfer from the ultimate sulfur donor to the substrate, (ii) some sulfur carrier proteins have multiple roles that deliver sulfur to a variety of cofactors and nucleosides, and (iii) the sulfur carriers vary significantly between different organisms. The bacterial s⁴U8 and s²U34 biosynthetic pathways have mainly been studied in Escherichia coli and Salmonella enterica, and they depend on persulfide group (R-S-SH) carriers for sulfur transfer (18). The first step of both pathways is to derive the sulfur element from free l-cysteine by cysteine desulfurases (IscS in E. coli), generating an initial protein-bound persulfide on an active site Cys residue in cysteine desulfurases (18). In E. coli, the persulfide of IscS can then be directly transferred to ThiI that catalyzes s⁴U8 formation or indirectly transferred to the s²U34 formation enzyme MnmA through a sulfur relay chain composed of multiple intermediate persulfide carriers (TusA, TusBCD complex, and TusE) (2, 19). Both E. coli ThiI and MnmA contain a PP-loop motif and two active site Cys residues critical for activities (Fig. 1). The PP-loop binds ATP that is consumed to activate the target uridine by adenylation (Fig. 1A). The first catalytic Cys (Cys456 in E. coli ThiI and Cys199 in E. coli MnmA) receives the persulfide from IscS, and subsequently the ThiI- and MnmA-persulfide donate the sulfur to thiolate the activated C4 atom of U8 and C2 atom of U34, respectively (20, 21). The second catalytic Cys (Cys344 in E. coli ThiI and Cys102 in E. coli MnmA) is proposed to form a disulfide bond with the first Cys, assisting sulfur release (20–22). The disulfide bond then needs to be reduced by a reductant before the next catalytic cycle. Notably, many bacterial ThiI homologs lack the rhodanese homology domain (RHD) (Fig. 1B) that contains the persulfide forming Cys (23, 24); therefore, the sulfur transfer mechanism of ThiI without RHD remains unclear.

Fig. 1.

The reactions (A) and domain structures (B) of s⁴U8 synthetase (ThiI), s²U34 synthetase (MnmA or Ncs6/Ncs2 complex), and SepCysS. (A) The AMP and phosphate moieties are highlighted in green, and the sulfur atoms in the products are highlighted in red. (B) The PP-loop motif and putative catalytic site Cys residues (with amino acid residue numbers indicated above) are colored in green and red, respectively. The domain structures of ThiI, MnmA, Ncs6, and SepCysS are based on the crystal structures of Bacillus anthracis ThiI (60), E. coli MnmA (21), Pyrococcus horikoshii TtuA (56), and Archaeoglobus fulgidus SepCysS (35), respectively. Abbreviations: Ado, adenosine; Ec, E. coli; Mmp, M. maripaludis; Sc, Saccharomyces cerevisiae; Mj, Methanocaldococcus jannaschii; NFLD, N-terminal ferredoxin-like domain; THUMP, thiouridine synthases, methylases and pseudouridine synthases; RHD, rhodanese homology domain; PLP, pyridoxal-5′-phosphate.

The mechanism by which sulfur is incorporated into s²U34 in eukaryotic cytosols differs greatly from that in bacteria. Initiated by Nfs1 (a cysteine desulfurase), the sulfur flow is directed through multiple persulfide carriers to a ubiquitin-related modifier (Urm1), generating a thiocarboxylate group on the carboxyl-terminus of Urm1 (10, 25). This thiocarboxylate may be a sulfur donor for s²U34 formation. The final step to activate and thiolate the C2 atom of U34 is catalyzed by an enzyme complex—designated as Ncs6/Ncs2 in yeast, Ctu1/Ctu2 in nematode, and ATPBD3/CTU2 in human (11, 25, 26)―in replacement of bacterial MnmA. Ncs6 has a PP-loop motif and three conserved Cys residues (two from a CXXC motif) in its putative catalytic domain (Fig. 1B). The PP-loop is presumably used for ATP binding to adenylate U34, resembling the reaction schemes of bacterial ThiI and MnmA; however, the sulfur transfer mechanism of Ncs6 is not known. Interestingly, the cytosolic Ncs6/Urm1 pathway is dependent on the Fe-S cluster assembly machineries (27), suggesting that it requires unidentified Fe-S proteins.

The archaeal s²U34 biosynthetic pathway is proposed to resemble the eukaryotic cytosolic Ncs6/Urm1 pathway. This is based on the observations that: (i) the ncs6 gene homologs are widespread in archaeal genomes (23); (ii) deletion of the ncs6 homolog (ncsA) in Haloferax volcanii resulted in only nonthiolated tRNA^Lys_UUU (28); (iii) Ncs6 homologs form complexes with the ubiquitin-like small archaeal modifier protein (SAMP), which has high structural homology to Urm1, in H. volcanii (29) and in Methanococcus maripaludis (30); and (iv) deletion of either samp2 or ubaA (encoding an E1-like protein that activates SAMPs) in H. volcanii eliminated thiolated tRNA^Lys_UUU (31). These findings suggest that both Ncs6 and the activated ubiquitin-like SAMP are required for s²U34 formation in archaea.

The archaeal s⁴U8 biosynthesis most likely resembles the bacterial ThiI pathway because ThiI is widely distributed in archaea and deletion of the thiI gene in M. maripaludis eliminated s⁴U in tRNAs (32). However, the E. coli s⁴U biosynthesis mechanism cannot fully explain the archaeal process because the gene encoding a cysteine desulfurase is missing in many sequenced archaeal genomes (23, 33) and most archaeal ThiI homologs lack the RHD essential to transfer sulfur. Similar to Ncs6, the methanogenic archaeal ThiI homologs have three conserved Cys residues (two from a CXXC motif) in the putative catalytic domain (Fig. 1B). A single mutation of any of the three Cys abolished M. maripaludis ThiI activity (32), demonstrating that all three Cys residues are crucial. This three-conserved Cys pattern is also present in Sep-tRNA:Cys-tRNA synthase (SepCysS), which catalyzes tRNA-dependent Cys biosynthesis in methanogenic archaea (Fig. 1) (34, 35). Mutational studies showed that the three Cys residues in SepCysS are all critical for activity (36, 37). These findings imply that the three proteins―Ncs6 (in eukaryotes and archaea), ThiI (in methanogenic archaea), and SepCysS (in methanogenic archaea)―have similar but unknown catalytic mechanisms dependent on three conserved Cys residues. Here we present evidence that these Cys residues coordinate a [3Fe-4S] cluster essential for tRNA thiolation.

Results

ThiI, SepCysS, and the Ncs6 Homolog in Methanogenic Archaea Contain a [3Fe-4S] Cluster.

The His₆-tagged M. maripaludis ThiI (locus tag: MMP1354), the M. maripaludis Ncs6 homolog (locus tag: MMP1356), and Methanocaldococcus jannaschii SepCysS (locus tag: MJ1678) proteins were recombinantly produced in E. coli and purified under anoxic conditions. The purified proteins were brownish in color (Fig. 2, Insets), exhibiting a broad band at around 420 nm in UV-visible absorption spectra (Fig. 2). Addition of 5 mM sodium dithionite (DTH) partially bleached the protein color and decreased the UV-visible absorption. Chemical analysis of Fe content indicated that the proteins contained 2.8 ± 0.2 Fe per protomer of M. maripaludis ThiI, 2.7 ± 0.3 Fe per protomer of M. maripaludis Ncs6, and 1.2 ± 0.5 Fe per protomer of M. jannaschii SepCysS. When any of the three conserved Cys was altered to Ala (C265, C268, and C348 in MMP1354; C142, C145, C233 in MMP1356; C64, C67, and C272 in MJ1678), the proteins were colorless and contained no detectable Fe (<0.1 Fe per protomer). These spectral properties and the presence of substantial amounts of associated Fe in wild-type proteins suggest that they contain Fe-S clusters coordinated by three conserved Cys residues.

Fig. 2.

UV-visible spectra of anoxically purified proteins in the as-purified (red) and after DTH reduced (blue) states. The His₆-tagged (A) M. maripaludis ThiI (330 µM), (B) M. maripaludis Ncs6 (540 µM), and (C) M. jannaschii SepCysS (670 µM) were produced and purified from E. coli. (Insets) The as-purified proteins were brownish in color; DTH reduction partially bleached the colors.

To confirm the presence of an Fe-S cluster and determine the cluster type, we used electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The EPR spectra of all three as-purified (following affinity purification steps inside an anaerobic chamber without addition of a reducing agent) proteins measured at 7 K displayed a peak centered at g_av = 2.00 (Fig. 3A and Table S1), characteristic of a cuboidal [3Fe-4S]¹⁺ cluster (S_tol = 1/2) containing three Fe³⁺ ions (38). After treatment with 5 mM DTH, all proteins were EPR-silent in perpendicular-mode measurement, consistent with a one-electron reduction of the cluster to the high spin [3Fe-4S]⁰ state (S_tot = 2) resulting from an Fe³⁺ ion and a mixed-valent (Fe^2.5+)₂ pair (38). Attempts to reoxidize the cluster in M. maripaludis ThiI by treatment with 2 mM potassium ferricyanide or by exposure to air were unsuccessful, resulting in precipitated protein and no significant EPR signal. The zero-field ⁵⁷Fe Mössbauer spectrum of the as-purified M. maripaludis ThiI recorded at 80 K displayed one quadrupole doublet with an isomer shift δ = 0.28 mm⋅s⁻¹ and a quadrupole splitting |∆E_Q| = 0.63 mm⋅s⁻¹ (Fig. 3B). These values indicate a [3Fe-4S]¹⁺ cluster with all Fe³⁺ ions (39). After reduction with 5 mM DTH, the spectrum was best fit to two quadrupole doublets in a 2:1 ratio (Fig. 3B). The more intense doublet with δ = 0.49 mm⋅s⁻¹ and |∆E_Q| = 1.08 mm⋅s⁻¹ corresponds to an (Fe^2.5+)₂ pair, and the less intense doublet with δ = 0.35 mm⋅s⁻¹ and |∆E_Q| = 1.13 mm⋅s⁻¹ corresponds to an Fe³⁺ site (Fig. 3B). These values agree with a reduced [3Fe-4S]⁰. Collectively, the EPR and Mössbauer spectra indicate that the as-purified proteins harbor an oxidized [3Fe-4S]¹⁺ cluster that can be completely reduced to [3Fe-4S]⁰ with DTH.

Fig. 3.

EPR and Mössbauer spectra of anoxically purified proteins. (A) The X-band EPR spectra of M. maripaludis ThiI (530 µM), M. maripaludis Ncs6 (540 µM), and M. jannaschii SepCysS (670 µM) in the as-purified (red) and DTH reduced (blue) states. The experimental conditions are as follows: microwave power, 2 μW for M. maripaludis ThiI, 200 μW for M. maripaludis Ncs6, 100 μW for M. jannaschii SepCysS; microwave frequency, 9.4 GHz; modulation amplitude, 10 G; temperature, 7K. (B) The zero-field ⁵⁷Fe Mössbauer spectra of the as-purified (Upper) and DTH reduced (Lower) M. maripaludis ThiI (180 µM). The experimental data (gray dots) were recorded at 80 K. (Upper) The best fit quadrupole doublet (red trace) has δ = 0.28 mm⋅s⁻¹ and |ΔE_Q|= 0.63 mm⋅s⁻¹. (Lower) The best fit (red trace) is a sum of two quadrupole doublets: the major component with δ = 0.49 mm⋅s⁻¹ and |ΔE_Q|= 1.08 mm⋅s⁻¹ (green) and the minor component with δ = 0.35 mm⋅s⁻¹ and |ΔE_Q| = 1.13 mm⋅s⁻¹ (blue). About 5% impurity is not included in the fit with δ = 0.71 mm⋅s⁻¹ and |ΔE_Q| = 3.04 mm⋅s⁻¹ indicating an Fe²⁺ species.

Table S1.

EPR conditions and simulation values

			g				g-Strain
Spectrum	ν, GHz	T, K	x	y	z	Average	x	y	z	Average
Fig. 3A (Top)	9.3883	7.5	1.9762	2.0187	2.0067	2.0005	0.0421	0.0050	0.0127	0.0199
Fig. 3A (Middle)	9.3851	7.1	1.9890	2.0168	2.0065	2.0041	0.0333	0.0066	0.0125	0.0175
Fig. 3A (Bottom)	9.3870	7.4	1.9789	2.0108	2.0072	1.9990	0.0379	0.0046	0.0124	0.0183
Fig. 5C	9.3848	8.8	1.9912	2.0222	2.0102	2.0078	0.0364	0.0045	0.0116	0.0175
Fig. 5D (Upper)	9.3859	7.2	1.9755	2.0216	2.0094	2.0022	0.0439	0.0052	0.0122	0.0204
Fig. 5D (Lower)	9.3881	7.6	1.9834	2.0206	2.0072	2.0037	0.0409	0.0058	0.0133	0.0200
Fig. S1B	9.3892	8.9	1.9931	2.0222	2.0108	2.0087	0.0353	0.0047	0.0120	0.0173

To verify that the recombinant protein produced in E. coli has the same cluster as the one in archaea, the Strep-tagged M. maripaludis ThiI was produced and anoxically purified from its natural host M. maripaludis. The as-purified protein contained 2.9 ± 0.1 Fe per protomer and exhibited identical features in the UV-visible (Fig. S1A) and EPR (Fig. S1B and Table S1) spectra compared with the protein purified from E. coli. These results suggest that M. maripaludis ThiI contains a [3Fe-4S] cluster irrespective to our tested expression hosts.

Fig. S1.

Spectroscopic characterization of Strep-tagged M. maripaludis (Mmp) ThiI produced and anoxically purified from M. maripaludis in the as-purified (red) and 5 mM DTH reduced (blue) states. (A) UV-visible spectra of 190 µM M. maripaludis ThiI. (B) The X-band EPR spectra was recorded at the following conditions: microwave power, 20 μW; microwave frequency, 9.4 GHz; modulation amplitude, 10 G; temperature, 9K.

Eukaryotic Ncs6 Contains a [3Fe-4S] Cluster.

The Saccharomyces cerevisiae Ncs6 and its human homolog (designated as CTU1 or ATPBD3) share ∼30% sequence identity with the M. maripaludis Ncs6 homolog. To characterize the eukaryotic protein, the maltose binding protein (MBP)-tagged human CTU1 was produced in E. coli and purified under anoxic conditions. The as-purified protein contained 1.0 ± 0.1 Fe per protomer and exhibited an absorption peak around 420 nm in the UV-visible spectrum (Fig. 4A). Furthermore, the ⁵⁷Fe Mössbauer spectrum of the as-purified human CTU1 recorded at 80 K displayed one quadrupole doublet with δ = 0.29 mm⋅s⁻¹ and |∆E_Q| = 0.58 mm⋅s⁻¹, consistent with the signal of an oxidized [3Fe-4S]¹⁺ cluster (Fig. 4B). These results suggest that the eukaryotic cytosolic Ncs6 has a [3Fe-4S] cluster similar to that in archaeal proteins.

Fig. 4.

Spectroscopic characterization of the recombinant MBP-tagged human CTU1. (A) UV-visible spectra of anoxically purified protein (420 µM) in the as-purified (red) and after DTH reduced (blue) states. (B) The zero-field ⁵⁷Fe Mössbauer spectrum of the as-purified CTU1 (420 µM). The experimental data (gray dots) were recorded at 80 K and fit to one quadrupole doublet with δ = 0.29 mm⋅s⁻¹ and |ΔE_Q| = 0.58 mm⋅s⁻¹ (red trace).

The [3Fe-4S] Cluster Is Essential for tRNA Thiolation Activity.

The importance of the Fe-S cluster for M. maripaludis ThiI enzyme activity was examined using [(N-acryloylamino)phenyl] mercuric chloride (APM) gel electrophoresis, a method that detects sulfur modifications in RNAs (40). Cysteine, thiosulfate, thiophosphate, and sulfide were previously tested as possible sulfur sources, but only sulfide supported the formation of thiolated tRNA exhibiting retarded migration in APM gels (32). The K_M of Na₂S (∼1 mM) (32) is close to the estimated intracellular concentrations of free sulfide in methanococci (∼1–3 mM) (41), suggesting that sulfide is a physiologically relevant sulfur donor. In this study, the anoxically purified M. maripaludis ThiI, which contained a [3Fe-4S] cluster, was active to produce thiolated tRNA when using M. jannaschii tRNA^Cys, Na₂S, and ATP as the substrates (Fig. 5A). When 5 μM of as-purified M. maripaludis ThiI was used in this assay, about 18 μM of M. jannaschii tRNA^Cys was thiolated according to band intensities (Fig. 5F); this finding suggests that M. maripaludis ThiI can catalyze multiple turnovers using exogenous sulfide. On the other hand, after incubation with 10 mM EDTA in the presence of 10 mM DTH, M. maripaludis ThiI became colorless and lost its cluster (<0.1 Fe per protomer). This apo-protein was inactive to produce thiolated tRNA (Fig. 5A), indicating that the Fe-S cluster is required for the tRNA thiolation activity of M. maripaludis ThiI.

Fig. 5.

The [3Fe-4S] cluster in M. maripaludis ThiI is essential for the tRNA thiolation activity. (A) The tRNA thiolation activity of the as-purified (Left), EDTA-treated (Center), and in vitro reconstituted M. maripaludis ThiI (Right) was analyzed with the APM-retardation gel electrophoresis assay. The protein (1 μM) was incubated with the unmodified M. jannaschii tRNA^Cys transcript (20 μM) together with 2 mM ATP and 5 mM Na₂S for 0, 5, 15, 30, or 60 min. The relative positions of unmodified and thiolated tRNAs are labeled. (B) The UV-visible spectra of EDTA-treated (gray) and reconstituted (red) M. maripaludis ThiI. (C) The X-band EPR spectrum of the reconstituted M. maripaludis ThiI recorded at microwave power, 20 μW; microwave frequency, 9.4 GHz; modulation amplitude, 10 G; temperature, 9 K. (D) The X-band EPR spectra of the as-purified M. maripaludis ThiI (280 μM) incubated with 5 mM Na₂S (Upper) or 2 mM ATP + 200 μM tRNA^Cys (Lower) recorded at microwave power, 20 μW; microwave frequency, 9.4 GHz; modulation amplitude, 10 G; temperature, 7 K. (E) The zero-field ⁵⁷Fe Mössbauer spectra of the as-purified M. maripaludis ThiI (340 µM) incubated with 5 mM Na₂S. The experimental data (gray dots) were recorded at 80 K. The best fit (red trace) is a sum of two quadrupole doublets―one with δ = 0.43 mm⋅s⁻¹ and |ΔE_Q|= 1.14 mm⋅s⁻¹ (blue) and the other one with δ = 0.30 mm⋅s⁻¹ and |ΔE_Q|= 0.60 mm⋅s⁻¹ (green)―in a 1:1 ratio. (F) The tRNA thiolation activity of as-purified M. maripaludis ThiI (5 μM) reacted with the unmodified M. jannaschii tRNA^Cys transcript (20 μM), 2 mM ATP, and 5 mM Na₂S (Left), 5 mM DTH (Center), or 5 mM Na₂S + 5 mM DTH (Right).

To reactivate the apo-protein, it was incubated with excess amounts of Fe²⁺ and sulfide in the presence of DTT. This treatment resulted in brownish-colored protein, showing a broad band at around 420 nm in the UV-visible absorption spectrum similar to the as-purified protein (Fig. 5B). The EPR spectrum displayed a peak centered at g_av = 2.01 (Fig. 5C and Table S1), demonstrating the successful reconstitution of an oxidized [3Fe-4S]¹⁺ cluster. The chemical analysis of Fe content agreed with the spin quantification of the EPR signal, indicating that ∼50% of the total protein turned into the holo-form after reconstitution. As expected, the in vitro reconstituted cluster restored the tRNA thiolation activity of M. maripaludis ThiI (Fig. 5A).

To investigate the active form of the cluster during reaction, M. maripaludis ThiI was incubated with the substrates (Na₂S or ATP + tRNA^Cys) and monitored by EPR spectroscopy. None of the substrates altered the g_av (Fig. 5D), suggesting that the cluster type at least partially remained as [3Fe-4S]¹⁺ upon substrate binding. In comparison with the as-purified protein, the spin quantification of the EPR signal was not significantly changed with the addition of ATP + tRNA^Cys but decreased with Na₂S. One possibility is that Na₂S partially reduced the [3Fe-4S]¹⁺ cluster to the EPR-silent [3Fe-4S]⁰ state. In agreement with the reduction, the zero-field ⁵⁷Fe Mössbauer spectrum of M. maripaludis ThiI incubated with Na₂S was best fit to the sum of two quadrupole doublets in a 1:1 ratio (Fig. 5E). One doublet with δ = 0.43 mm⋅s⁻¹ and |∆E_Q| = 1.14 mm⋅s⁻¹ corresponds to Fe^2.5+, and the other doublet with δ = 0.30 mm⋅s⁻¹ and |∆E_Q| = 0.60 mm⋅s⁻¹ corresponds to Fe³⁺ (Fig. 5E); this spectrum suggests a mixture of 25% of [3Fe-4S]¹⁺ and 75% of [3Fe-4S]⁰ after incubation with Na₂S. To further investigate whether the reduced state is active, we tested the DTH-treated M. maripaludis ThiI containing only the reduced cluster. This completely reduced enzyme showed similar activity to the as-purified protein containing the oxidized cluster (Fig. 5F). These observations suggest that the enzyme can catalyze the thiolation reaction irrespective of the initial redox state of the cluster, although we cannot eliminate the possibility that the initially oxidized cluster needs to be reduced by Na₂S for activity. Furthermore, the reduced enzyme was unable to produce thiolated tRNA without Na₂S (Fig. 5F), suggesting that Na₂S is required as an exogenous sulfur source rather than a reductant under this experimental condition.

Discussion

Taking these data together, we demonstrate by UV-visible absorbance, EPR, and Mössbauer spectroscopies that the archaeal and eukaryotic Ncs6 homologs as well as the methanogenic archaeal ThiI and SepCysS are all [3Fe-4S] cluster-containing enzymes. Despite lacking significant sequence homology, these three proteins all have three conserved Cys residues (two from a CXXC motif) in their putative catalytic domains. A single mutation of any of the three Cys removed protein-bound Fe, suggesting that these Cys residues chelate the Fe-S cluster. A trisulfide linkage was previously detected in the CXXC motif by mass spectrometry analyses of M. maripaludis ThiI, M. maripaludis Ncs6 homolog, and M. jannaschii SepCysS (30, 32, 36); however, the sulfane sulfur (S⁰) carried by Cys may come from oxidation of an Fe-S cluster, which was similarly observed upon oxidative decomposition of a spinach ferredoxin (42) and aconitase (43). The three conserved Cys residues are essential for in vivo activities of M. maripaludis ThiI (32) and M. jannaschii SepCysS (36). Similarly, the corresponding CXXC motif is required for the Ncs6 activity in yeast (11). In this study, we reveal that the [3Fe-4S] cluster is indispensable for the tRNA thiolation activity of M. maripaludis ThiI; this suggests that the critical role of these conserved Cys residues is Fe-S cluster coordination. Overall, the same essential Cys pattern and cluster type discovered in ThiI, Nsc6, and SepCysS suggest that they have a common reaction mechanism dependent on a [3Fe-4S] cluster. This finding now explains why previous genetic studies showed that the biosynthesis of s²U in eukaryotic cytosolic tRNAs requires the Fe-S cluster assembly machinery (27).

The reaction mechanism of a [3Fe-4S] cluster-dependent thiolation remains to be explored. The [3Fe-4S] clusters are less common than the cubic [4Fe-4S] clusters in natural proteins. A few cases of [3Fe-4S]-containing proteins include [NiFe] hydrogenase (44), succinate dehydrogenase (45), and nitrate reductase (46), in all of which the [3Fe-4S] clusters have been proposed to transfer electrons. We propose three thoughts of the role of the [3Fe-4S] clusters in tRNA thiolation reactions. (i) The cluster is possibly required for binding to a substrate such as tRNA, ATP, or an adenylated intermediate. For example, the [4Fe-4S] clusters in E. coli RlmD/RumA (47) and Thermotoga maritima tryptophanyl-tRNA synthetase (TrpRS) (48) are proposed to be involved in RNA substrate recognition but not directly in catalysis. (ii) The cluster may be a direct sulfur donor, providing a sulfur atom that is inserted to the substrate. For example, a [2Fe-2S] cluster in biotin synthase has been proposed to donate a sulfur atom into the biotin thiophane ring; the cluster is killed during the reaction (49). To explain that exogenous Na₂S is required for the M. maripaludis ThiI reaction and M. maripaludis ThiI can have multiple turnovers, the [3Fe-4S] cluster needs to be regenerated from sulfide during catalysis. (iii) The cluster may be responsible for sulfur transfer by ligating to an activated sulfur species. For example, the methylthiotransferases MiaB, which catalyzes the conversion of i⁶A to ms²i⁶A in tRNAs, carries a polysulfide bridge connecting its two [4Fe-4S] clusters (50). This polysulfide is likely derived from an exogenous sulfur source and used as the sulfur donor to generate ms²i⁶A. Similar to the proposed MiaB reaction mechanism, an exogenous sulfur may be ligated to an Fe ion of the [3Fe-4S] cluster in Nsc6, ThiI, and SepCysS and then transferred to their substrates. The Fe ion, when ligated to an activated sulfur species, may dissociate from its Cys residue ligand or still link to the residue as a Cys persulfide ligand (51).

The Ncs6 sequence is most closely related to TtuA and TtcA, belonging to the TtcA/TtuA family. TtuA catalyzes the biosynthesis of 2-thioribothymidine (s²T) at position 54 in thermophilic bacterial and archaeal tRNAs (52, 53), whereas TtcA catalyzes s²C formation at position 32 in bacterial and archaeal tRNAs (54). The TtcA sequence has two CXXC motifs, and a recent study of E. coli TtcA showed that this protein possesses a [4Fe-4S] cluster that is crucial for the thiolation activity (55). Although the reaction mechanism remains unclear, the [4Fe-4S] cluster is suggested to coordinate a sulfide (–SH) group that is the proximal sulfur donor to generate s²C (55). In contrast, both Ncs6 and TtuA contain five CXXC(H) motifs, which are characteristics of the group II TtcA family (54). A crystal structure of TtuA showed that TtuA has two Zn finger domains (each with two CXXC/H motifs) and a putative catalytic domain (with a PP-loop motif, a CXXC motif, and an additional conserved Cys) (56). Mutational analyses revealed that the three Cys residues in the putative catalytic domain are essential for TtuA activity (56). Although the solved crystal structure of TtuA did not show the presence of a cluster, the same requirement of Cys residues in Ncs6 and TtuA suggests that TtuA may also have an Fe-S cluster coordinated by the three critical Cys residues. In this scenario, the requirement of an Fe-S cluster is a common feature of the TtcA/TtuA family that is used for tRNA thiolation by all three domains of life.

Materials and Methods

Production and Anoxic Purification of His₆-Tagged M. maripaludis ThiI, M. maripaludis Ncs6, and M. jannaschii SepCysS.

The M. maripaludis thiI (locus tag: MMP1354), M. maripaludis ncs6 (locus tag: MMP1356), and M. jannaschii pscS (locus tag: MJ1678) genes were cloned into the pQE2 (with an N-terminal His₆-tag), pDCH (with a C-terminal His₆-tag), and pET15b (with an N-terminal His₆-tag) vectors, respectively. For expression, the vectors were transformed into the E. coli Rosetta 2(DE3) strain (Novagen). The details of protein production and purification are in SI Materials and Methods.

Production and Anoxic Purification of Strep-Tagged M. maripaludis ThiI from M. maripaludis.

The M. maripaludis thiI gene (locus tag: MMP1354) was cloned into the pAW42 vector with an N-terminal Strep-tag (WSHPQFEK) and transformed into the M. maripaludis strain S0001 for expression. The details of culture condition, transformation, expression, and protein purification are in SI Materials and Methods.

Production and Anoxic Purification of MBP-Tagged ATPBD3.

The codon optimized Homo sapiens gene ATPBD3 (CTU1) was cloned into the pMA-c5x vector (New England Biolabs) with an N-terminal MBP-tag. The expression vectors were transformed into the E. coli BL21(DE3) strain. The details of protein production and purification are in SI Materials and Methods.

Analytical and Spectroscopic Measurements.

All analytical analyses were performed in triplicate. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce). Iron was quantified by using the Quantichrom Iron Assay Kit (BioAssay Systems). UV-visible absorption spectra were recorded on a Nanodrop 2000c spectrometer with samples in quartz cuvettes (optic path = 1 cm) closed with rubber stoppers under anoxic conditions.

X-band EPR spectra were recorded at 7–9 K on a Bruker ELEXSYS E500 spectrometer equipped with a SHQ resonator and Oxford ESR-900 helium flow cryostat. Multiple microwave powers were tested so that resonances were quantified under nonsaturating conditions. The g values (Table S1) were determined by simulating spectra using EasySpin 5.0.5 (57). Mössbauer spectra were recorded at 80 K on a SeeCo Mössbauer spectrometer with alternating acceleration and a 0.07 mT magnetic field. The isomer shifts were referenced to iron metal at 298 K. Sample temperature was maintained at 80 K with a Janis Research Company cryostat. The spectra were simulated and fit using WMoss (SeeCo) with Lorentzian doublets. The fits are anisotropic as a result of the clusters’ paramagnetism affecting the relaxation time of the transitions (58, 59).

In Vitro Reconstitution of Fe-S Cluster and Enzymatic Assay.

All following steps were performed under anoxic conditions. To prepare apo-protein, the purified M. maripaludis ThiI was treated with 10 mM EDTA and 10 mM sodium DTH at 4 °C for 4 h. The apo-protein was buffer-exchanged using a PD MiniTrap G-25 column (GE Healthcare) pre-equilibrated with 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, and 25% (vol/vol) glycerol. To reconstitute the Fe-S cluster, the protein was first incubated with 5 mM DTT at room temperature for 1 h. Then a 10-fold molar excess of Na₂S followed by an 8-fold molar excess of Fe(NH₄)₂(SO₄)₂ was added. The mixture was incubated at room temperature for 1 h or at 4 °C overnight. The excess reagents were then removed by passing through a PD MiniTrap G-25 column (GE Healthcare) pre-equilibrated with 50 mM sodium Hepes (pH 7.5) and 0.3 M NaCl. After concentrating with an Amicon Ultra centrifugal filter (30-kDa molecular weight cut-off), the reconstituted protein was supplemented with glycerol [final concentration 30% (vol/vol)].

The in vitro tRNA thiolation reaction catalyzed by M. maripaludis ThiI was performed under anoxic conditions as described (32). ATP (final concentration 2 mM), M. jannaschii tRNA^Cys transcript (final concentration 20 µM), and freshly prepared Na₂S (final concentration 5 mM) were added to the as-purified, EDTA-treated, or cluster reconstituted M. maripaludis ThiI (1 μM) to initiate the reaction. To observe time-dependence, 50-μL reactions in buffer containing 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl₂ was carried out at 37 °C for 5–60 min. At each time point, 6 μL aliquots were mixed with 6 μL formamide loading dye to quench the reaction. The mixture was analyzed by APM-retardation gel electrophoresis as described previously (32). The relative positions of unmodified and thiolated tRNA^Cys were compared with those reported previously (32).

SI Materials and Methods

Production and Anoxic Purification of His₆-Tagged Methanococcus maripaludis ThiI, M. maripaludis Ncs6, and Methanocaldococcus jannaschii SepCysS.

The M. maripaludis thiI (locus tag: MMP1354), M. maripaludis ncs6 (locus tag: MMP1356), and M. jannaschii pscS (locus tag: MJ1678) genes were cloned into the pQE2 (with an N-terminal His₆-tag), pDCH (with a C-terminal His₆-tag), pET15b (with an N-terminal His₆-tag) vectors, respectively. For expression, the vectors were transformed into the Escherichia coli Rosetta 2(DE3) strain (Novagen). The transformants were grown in 2 L LB medium at 37 °C with rotation at 200 rpm until OD₆₀₀ ∼ 0.6. Then the medium was supplemented with 200 μM ammonium ferric citrate, 30 μM l-Met, and 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) to induce overnight production at 25 °C with rotation at 110 rpm. For ⁵⁷Fe labeling, the cells were grown in 5 L M9 medium supplemented with 0.2% glucose (wt/vol) and 0.2% (wt/vol) casamino acids (Bacto) at 37 °C with rotation at 200 rpm until OD₆₀₀ ∼ 0.6. Then 100 μM ⁵⁷Fe dissolved in concentrated HCl, 30 μM l-Met, and 0.5 mM IPTG were added for overnight protein production at 25 °C with rotation at 110 rpm.

For protein purification, the harvested E. coli cells were transferred to an anaerobic chamber (Coy Laboratories) with an atmosphere of 95% (vol/vol) N₂ and 5% (vol/vol) H₂. All reagents and buffers were allowed to sit for enough time inside the chamber for complete deaeration. The cells were resuspended in 20 mL binding buffer containing 50 mM sodium Hepes at pH 7.5, 0.3 M NaCl, 5 mM MgCl₂, and 20 mM imidazole. Cells were lysed with 2 mL of 10× BugBuster (Novagen) in the presence of 0.5 mg/mL lysozyme, 10 U Benzonase (Sigma), and one pellet of cOmplete EDTA-free Protease Inhibitor Mixture (Roche). The cell lysate was centrifuged at 23,000 × g for 1 h at 4 °C. All following processes were carried out at room temperature. The clarified supernatant was applied to 2 mL of TALON Metal Affinity Resin (Clontech) equilibrated with the binding buffer. Proteins bound to the resin were washed with 40 mL binding buffer and then eluted with 10 mL elution buffer containing 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl₂, and 0.2 M imidazole. After concentrating with an Amicon Ultra centrifugal filter (30-kDa molecular weight cut-off), the purified protein was supplemented with glycerol [final concentration 30% (vol/vol)] and stored at –80 °C until use.

Production and Anoxic Purification of Strep-Tagged M. maripaludis ThiI from M. maripaludis.

The M. maripaludis thiI gene (locus tag: MMP1354) was cloned into the pAW42 vector (61) with an N-terminal Strep-tag (WSHPQFEK). The expression vector was transformed into the M. maripaludis strain S0001 (61) using the PEG method (62) to obtain the strain S795. For protein production, S795 was grown in 1.6 L McC (rich medium) with 2.5 μg/mL puromycin. The medium was reduced with 3 mM l-Cys, and before inoculation 3 mM sodium sulfide was added as the sulfur source. The culture was pressurized with 275 kPa of H₂:CO₂ (4:1, vol/vol) and incubated at 37 °C with rotation at 100 rpm until OD₆₀₀ ∼ 1.

All following steps were performed under anoxic conditions. The cells were harvested by centrifugation at 4,000 × g for 10 min at 4 °C and then resuspended in 10 mL binding buffer containing 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, and 5 mM MgCl₂. The cells were lysed by twice freezing (–80 °C) and thawing in the presence of one pellet of cOmplete EDTA-free Protease Inhibitor Mixture (Roche). DNA and RNA were digested with 10 U Benzonase (Sigma). The cell lysate was centrifuged at 23,000 × g for 30 min at 4 °C. The clarified supernatant was mixed with 4 mL of Strep-Tactin resin suspension (Qiagen) equilibrated with the binding buffer and incubated at 4 °C for 1 h with shaking. The mixture was then loaded into a 20 mL gravity flow column. Proteins bound to the resin were washed with 20 mL binding buffer and subsequently eluted with 10 mL elution buffer containing 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl₂, and 2.5 mM desthiobiotin. The concentrating and storage of purified protein was performed as the His-tagged proteins described above.

Production and Anoxic Purification of MBP-Tagged ATPBD3.

The codon optimized Homo sapiens gene ATPBD3 (CTU1) was cloned into the pMA-c5x vector (New England Biolabs) with an N-terminal MBP-tag. The expression vectors were transformed into the E. coli BL21(DE3) strain. The protein was produced using the same procedures as the His-tagged proteins described above, except that the overnight protein production was induced with 0.1 mM IPTG at 16 °C.

All following steps were performed under anoxic conditions. The cells were resuspended in 20 mL binding buffer containing 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl₂, and 10% (vol/vol) glycerol. Cells were lysed with 2 mL of 10× BugBuster (Novagen) in the presence of 0.5 mg/mL lysozyme, 10 U Benzonase (Sigma), and two pellets of cOmplete EDTA-free Protease Inhibitor Mixture (Roche). The cell lysate was centrifuged at 23,000 × g for 1 h at 4 °C. The clarified supernatant was applied to 2 mL of amylose resin (New England Biolabs) equilibrated with the binding buffer. Proteins bound to the resin were washed with 20 mL binding buffer and then eluted with 10 mL elution buffer containing 50 mM sodium Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl₂, 50 mM maltose, and 10% (vol/vol) glycerol. The concentrating and storage of purified protein was performed as the His-tagged proteins described above.