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J. Biol. Chem., Vol. 278, Issue 32, 29515-29524, August 8, 2003
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From the
Laboratoire de Chimie et Biochimie des
Centres Rédox Biologiques, Département Réponse et
Dynamique Cellulaires-Chimie Biologique (DRDC-CB), UMR 5047 Commissariat
à l'Energie Atomique/CNRS/Université Joseph Fourier,
Commissariat à l'Energie Atomique/Grenoble 17 avenue des Martyrs, 38054
Grenoble Cedex 09, France, and the Department of
Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens,
Georgia 30602
Received for publication, February 12, 2003 , and in revised form, May 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Escherichia coli four different thiolated nucleosides have been characterized. The best characterized biosynthetic pathway is that of 4-thiouridine, which is formed from uridine through a reaction catalyzed by the ThiI protein (10). The tRNA modification leading to 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37) involves a chemically intriguing and difficult aromatic C-H to C-S bond conversion, the mechanism of which has yet to be investigated. The modified nucleoside ms2i6A-37 is found at position 37, next to the anticodon on the 3' position in almost all eukaryotic and bacterial tRNAs that read codons beginning with U except tRNAI,V Ser (11). The postulated pathway for the synthesis of ms2i6A-37 and the proteins involved are shown in Fig. 1 (2, 12, 13).
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The biosynthesis of ms2i6A-37 requires at least two enzymatic activities, and the corresponding genetic loci are designated miaA and miaB. A third gene, miaC, has been postulated but has not been identified (Fig. 1). In E. coli, the first step of the biosynthesis of ms2i6A-37 is the addition of the isopentenyl group to the N-6 nitrogen of adenosine. This reaction is catalyzed by the well characterized tRNA-isopentenylpyrophosphate transferase enzyme, encoded by the miaA gene (14–16). The second step, which is dependent on iron, cysteine, S-adenosylmethionine (SAM), consists of both sulfur insertion and methylation at position 2 of the base moiety but it is still unknown whether each reaction is catalyzed by a single enzyme (MiaB+MiaC) or both by the same enzyme MiaB (17–19). However, tRNAs from mutant strains lacking a functional miaB gene have been shown to contain only i6A-37, the product of the first step of the pathway, suggesting that the MiaB protein is involved at least in C-S bond formation (20).
The main feature of MiaB protein sequences, which has emerged from the first sequence determination (20) and has been strongly substantiated when significant comparisons became possible (21), is the presence of highly conserved motifs. The N-terminal half of all MiaB enzymes contains a strictly conserved canonical cysteine triad CysXXXCysXXCys, which is also found in biotin and lipoate synthases. In these two enzymes, which also catalyze C-H to C-S bond conversion reactions, this motif was shown to provide cysteine ligands for a catalytically essential [4Fe-4S]+2/+1 cluster. Furthermore, it is the signature for a superfamily of iron-sulfur enzymes involved in a great variety of biosynthetic pathways and metabolic processes that function via radical mechanisms (22). Because we recently showed that MiaB from E. coli was an iron-sulfur protein with one [4Fe-4S] cluster per polypeptide chain and that the three cysteines of the CysXXXCysXXCys motif were required for activity (23), it is very likely that MiaB, biotin synthase, and lipoate synthase employ similar radical mechanisms for activation of sulfur and its insertion into their respective substrates.
Thermotoga maritima is a hyperthermophilic bacterium with an optimal growth temperature of 80 °C and is one of the deepest and most slowly evolving eubacterial lineage (24). The genomic sequence of T. maritima shows an ORF (TM0653) encoding a putative MiaB tRNA-methylthiotransferase. Because MiaB from E. coli proved quite difficult to manipulate and characterize and to gain more insight into the structure of the MiaB enzymes, we have extended our work on thiolation of tRNAs by investigating the MiaB protein from T. maritima. We report its purification and characterization, and show that it displays some properties (stability and homogeneity of the ironsulfur cluster) that have not been observed with other enzymes of the "radical-SAM" superfamily. Furthermore, we demonstrate that this protein is indeed a MiaB enzyme because its expression in an E. coli strain lacking its own miaB gene (20) results in the formation of ms2i6A. Finally, we show that the modified nucleoside ms2i6A is actually present in tRNA from T. maritima.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was used for routine DNA manipulations. E.
coli BL21CodonPlus(DE3)-RILTM (Stratagene), which contains extra
copies of genes encoding tRNA with codons rarely used in E. coli
(argU, ileY, and leuW tRNA genes) was used to produce the
recombinant protein. T. maritima (strain DSMZ3109) was a gift from
the laboratory of Professor Robert Huber (University of Regensburg,
Regensburg, Germany). E. coli strain TX3346 miaB– was
a gift of Professor Malcolm E. Winkler (University of Texas, Houston, TX).
General Procedures
All DNA manipulations were as described previously
(25). Enzymes,
oligonucleotides, and culture media were purchased from Invitrogen
(Cergy-Pontoise, France). T4 DNA ligase was from Promega, Inc. Bacterial
alkaline phosphatase and plasmid DNA purification kit, FlexiprepTM, were
from Amersham Biosciences. DNA fragments were extracted from agarose gel and
purified with the High Pure PCR product purification kit (Roche Molecular
Biochemicals). DNA sequencing was performed by Genome Express Co. (Grenoble,
France).
Cloning of the MiaB Gene and Construction of the Overexpressing
Plasmid
The TM0653 gene, encoding the MiaB protein named MiaBTm, was
identified from the GenBankTM data base using the BLAST search algorithm
(26). The open reading frame
was amplified by polymerase chain reaction (PCR)-based method using genomic
DNA of T. maritima as a template. The following primers were used:
5'-gggaggtcgcatATGagattttacataaag-3' (NdeI
site underlined, ATG codon in uppercase) hybridized to the noncoding strand at
the 5' terminus of the gene and
5'-gacaagggaagaaagcttgtccaccgtcgtg-3' (HindIII
site underlined) hybridized to the coding strand, 
25 bp after the TGA
stop codon. PCR was run on a Stratagene RoboCycler Gradient 40 machine as
follows. Genomic DNA (0.5–1 µg) was denatured for 4 min at 95 °C
in the presence of the primers (0.5 µM each). The Pwo
DNA polymerase (2 units) and deoxynucleotide mix (0.2 mM each) were
added, and 25 cycles (1 min at 95 °C, 1 min at 52 °C, 2 min at 72
°C) were then performed followed by a final 10-min elongation step at 72
°C. The PCR product was digested with NdeI and HindIII
and then ligated with T4 DNA ligase into the pT7-7 plasmid
previously digested with the same restriction enzymes. The cloned gene was
entirely sequenced to ensure that no error was introduced during PCR reaction.
The plasmid was then named pT7-MiaBTm.
Overexpression of the Recombinant Protein MiaBTm
The protein was overexpressed in E. coli
BL21CodonPlus(DE3)-RILTM. The transformation of competent cells was
carried out following the instructions of the manufacturer. Then a single
colony from a LB plate was transferred into 100 ml of LB medium supplemented
with ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml). The bacteria
were grown overnight at 37 °C, and 50 ml of this culture were used to
inoculate 10 liters of fresh LB medium supplemented with the same antibiotics.
Bacterial growth proceeds at 37 °C until A600 reached
0.4, the time at which protein expression was induced by adding 200
µM isopropyl-1-thio-
-D-galactopyranoside. Cells
were collected after 3 h of culture at 37 °C by centrifugation at 5000
x g at 10 °C, then resuspended in 50 mM Tris-Cl,
pH 8, containing 200 mM NaCl and stored at –70 °C until
use.
Purification of MiaBTm
The frozen cells were thawed, disrupted by sonication, and centrifuged at
220,000 x g at 4 °C for 90 min. The cell-free extracts were
heated at 75 °C for 15 min, and the denatured E. coli proteins
were eliminated by centrifugation at 14,000 x g at 4 °C for
15 min. The nucleic acids were partially eliminated by adding 0.04%
polyethyleneimine. The proteins present in the supernatant were precipitated
with 60% ammonium sulfate. The pellet was resuspended in 50 mM
Tris-Cl, pH 8; 50 mM Tris-Cl, pH 8, 1.5 M ammonium
sulfate was added to reach a final concentration of 1 M ammonium
sulfate. The solution was then loaded onto a butyl-Sepharose column previously
equilibrated with 50 mM Tris-Cl, pH 8, 1 M ammonium
sulfate. The column was washed extensively with the same buffer, and the
adsorbed proteins were eluted by a linear gradient from 1 to 0 M
ammonium sulfate. Fractions containing the protein were pooled and
concentrated in an Amicon cell fitted with a YM30 (Spectrapor) membrane. The
protein was then loaded on a Superdex 200 gel filtration column equilibrated
with 50 mM Tris-Cl, pH 8, 200 mM NaCl, 5 mM
DTT. The colored fractions that contained the protein were pooled, and DTT was
removed from the protein solution by using Centricon 30 devices (Amicon). The
pure protein was divided into aliquots and stored frozen at –70 °C
until use.
Aggregation State Analysis
Fast protein liquid chromatography gel filtration with a Superdex 200 at a
flow rate of 0.5 ml/min was used for size determination. The running buffer
was 50 mM Tris-Cl, pH 8, 200 mM NaCl. The column was
calibrated with a gel filtration calibration kit (calibration proteins II,
Roche Molecular Biochemicals).
Preparation of the Apoprotein
Protein-bound iron was removed by overnight exposure to EDTA (10
mM) under reducing conditions (10 mM sodium dithionite)
at 4 °C. After incubation, the colorless protein was loaded onto a
Sephadex NAP-25 gel filtration column equilibrated with 50 mM
Tris-Cl, pH 8, 200 mM NaCl. The apoMiaBTm was then washed and
concentrated with the same buffer using Centricon 30 devices (Amicon).
Reconstitution of the Iron-Sulfur Cluster of ApoMiaBTm
All the following procedures were carried out anaerobically inside a glove
box containing less than 2 ppm O2. The apoMiaBTm was treated with 2
mM DTT for 20 min and then a 6 M excess of
Na2S and FeCl3, from a solution containing DTT, were
added. The mixture was incubated for 3 h and chromatographed on a Sephadex
G-25 column. The dark brown fractions were collected and concentrated using an
Amicon cell.
Analytical Methods
Protein concentration was measured by the method of Bradford using bovine
serum albumin as a standard
(27) and also checked using a
Bicinchonic Acid Protein Kit Assay (Sigma) with the same standard. Iron was
determined by the method of Fish
(28), and inorganic sulfide
was quantified as described by Beinert
(29).
Analysis of T. maritima tRNA Nucleoside Composition by HPLC
Total tRNA from T. maritima (strain DSMZ3109) was prepared
essentially as described by Buck et al.
(30). tRNA samples were
digested to nucleosides by the method of Gehrke et al.
(31) by using nuclease P1 and
bacterial alkaline phosphatase. 50–100 µg of tRNA was loaded onto
LC-18 HPLC column (Vydac) connected to a HP-1100 HPLC system. The gradient
profile developed by Gehrke and Kuo was used to separate the different
nucleosides (31).
DNA and amino acid sequences were analyzed using the DNA Strider 1.3 software package. Sequence comparisons and homology searches implemented the BLAST program (26) at the NCBI server (www.ncbi.nlm.nih.gov). The amino acid sequences were aligned using ClustalW (32) at the EBI server (www.ebi.ac.uk/clustalW).
Spectroscopic Measurements
Light Absorption—UV-visible absorption spectra were recorded
with a Cary 1 Bio (Varian) spectrophotometer.
Electron Paramagnetic Resonance (EPR)—X-band EPR spectra were recorded on a Brucker Instruments ESP 300D spectrometer equipped with an Oxford Instruments ESR 900 flow cryostat (4.2–300 K). Spectra were quantified under non-saturating conditions by double integration against a 1 mM CuEDTA standard.
Resonance Raman (RR)—RR spectra were recorded using an
Instruments SA U1000 spectrometer fitted with a cooled RCA 31034
photo-multiplier tube with 90° scattering geometry. Spectra were recorded
digitally using photon-counting electronics, and improvements in
signal-to-noise were achieved by signal averaging multiple scans. Band
positions were calibrated using the excitation frequency and CCl4
and are accurate to ±1 cm–1. Lines from a
Coherent Sabre 10-W argon ion laser were used for excitation, and plasma lines
were removed using a Pellin Broca prism premonochromator. For each sample,
200 mW of incident laser power was used, and slit widths were adjusted
for each excitation wavelength to give 8.0 cm–1
spectral resolution. Scattering was collected from the surface of a frozen
18-µl droplet of sample using a custom designed anaerobic sample cell
(33) attached to the cold
finger of an Air Products Displex model CSA-202E closed cycle refrigerator.
This enables samples to be cooled down to 17 K, which facilitates improved
spectral resolution and prevents laser-induced sample degradation. A linear
ramp fluorescent background has been subtracted from each spectrum shown in
this work.
Variable Temperature Magnetic Circular Dichroism
(VTMCD)—VT-MCD measurements were made on samples containing 60%
(v/v) ethylene glycol in 1-mm cuvettes using a Jasco J-715 (180–1000 nm)
spectropolarimeter mated to an Oxford Instruments SM4000 split-coil
superconducting magnet. The experimental protocols for measuring anaerobic MCD
spectra over the temperature range 1.5–300 K with magnetic fields up to
6 T have been described elsewhere
(34,
35). The MCD intensities are
expressed as 
(LCP– RCP),
where LCP and RCP are the molar extinction
coefficients for the absorption of left and right circularly polarized light,
respectively.
Electrochemistry
Electrochemical experiments were performed in a closed three-electrode cell
as described previously (36).
The three-electrode design included a saturated Ag/AgCl reference
micro-electrode MI-401F from Microelectrode Inc, a platinum wire counter
electrode from EG&G Instruments, and a gold working electrode from
Radiometer. The three electrodes were connected to a EG&G Instruments
model 273A potentiostat/galvanostat controlled with model software. The gold
disc tip of the working electrode was hand-polished with
Al2O3 and cleaned with distilled water before each
analysis of sample. Reconstituted protein (200 µM) was prepared
under anaerobic conditions in 50 mM Tris-Cl, pH 8, containing 200
mM NaCl. The solution (10–20 µl) was transferred with a
Hamilton gas-tight syringe on the working electrode, and other electrodes were
positioned to contact the drop. The measurement was carried out anaerobically
at 25 °C, with a pulse height of 80 mV, a frequency of 20 Hz, and a
potential increment of 2 mV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100 amino acids), which is highly conserved, and in the case of E.
coli were shown to be also essential for activity
(20).2
Furthermore, MiaBTm shares with the other MiaB enzymes a sequence of
60–80 residues, in the C-terminal part, which is proposed to form a
domain reminiscent of the -barrel RNA-binding domains
(21). The five predicted
strands of this domain are marked with black boxes in
Fig. 2. This domain was named
TRAM (for TRM2 and MiaB) and is most likely
the domain of MiaB that binds the tRNA substrate. MiaBTm thus consists of 443
amino acids, and the molecular mass of the apoprotein as calculated from the
sequence is 50,710 Da, comparable with that of the E. coli enzyme
(53,600 Da).
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Analysis of tRNA Nucleoside Composition from T. maritima by HPLC—Total tRNA from T. maritima cells was prepared and digested with nuclease P1 and phosphatase, and the released nucleosides were analyzed by HPLC. The elution profile of a tRNA hydrolysate is shown in Fig. 3. A nucleoside with a retention time (84 min) corresponding to the one of ms2i6A was detected. Moreover, this modified nucleoside has a UV-visible spectrum (Fig. 3, inset) identical to that of ms2i6A from E. coli (31). Thus, we conclude that the tRNA from T. maritima contains the ms2i6A modified nucleoside and that as a consequence these bacteria have a MiaB enzyme for introduction of the sulfur atom.
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Cloning, Overexpression, and Purification of the MiaB Protein from T.
maritima—The open reading frame (ORF) TM0653 was amplified
from the genomic DNA by PCR reaction technology and cloned into a
pT7-7 vector. The E. coli BL21CodonPlus(DE3)-RILTM
strain was transformed with the resulting expression vector, named
pT7-MiaBTm, which establishes control over TM0653 gene
expression with the T7 RNA polymerase promoter.
Isopropyl-1-thio--D-galactopyranoside induction of the
transformed E. coli cells resulted in the over-production of a
protein that migrates at 50 kDa on SDS gels
(Fig. 4, lane 2) and
was found mainly in the soluble fraction of cell free extracts
(Fig. 4, lane 3).
After the final step of purification, the purity was evaluated by SDS-PAGE to
be over 95% (Fig. 4, lane
7). Typical yields were 40 mg of pure protein from a 10-liter
culture. The apparent molecular mass of MiaBTm determined by analytical gel
filtration chromatography (Superdex 200) showed that the protein is mainly a
monomer in solution (data not shown). Indeed, whereas the pure protein eluted
in different peaks corresponding to multimeric, dimeric, and monomeric states,
the latter was by far the major one. When 5 mM DTT was added in the
protein solution and in the eluting buffer, only the monomer was observed.
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The TM0653 Gene from T. maritima Complements the ms2 Deficiency of an E. coli miaB Mutant Strain—The functionality of the MiaB protein from T. maritima was assayed in vivo using the miaB– TX3346 E. coli strain lacking a functional miaB gene (20). This strain was transformed with the plasmid pT7-MiaBTm. Cells were first cultivated at 37 °C until A600 reached 0.8. tRNAs were then isolated, and their modified nucleoside content was analyzed by HPLC, as described (31). Under these conditions tRNAs from the control strain (TX3346) as well as the ones from the pT7-miaBTm transformed strain showed an accumulation of the i6A-37 with no evidence for the presence of ms2i6A-37 (Fig. 5A). In a second experiment the control and the transformed cells were grown at 37 °C until A600 reached 0.4 and then shifted to 45 °C for 3 h. Whereas the tRNAs from the control strain still showed only an accumulation of i6A-37, those from the MiaBTm-expressing strain showed both i6A-37 and ms2i6A-37 (Fig. 5B). These results demonstrate that the product of the TM0653 gene is a functional MiaB enzyme even for modification of E. coli tRNAs, catalyzing the conversion of i6A-37 to ms2i6A-37 in vivo. However, as expected for a thermophilic enzyme, this reaction requires a temperature higher than 37 °C. The fact that the enzyme is not working at the optimal temperature (80 °C) explains why the conversion of i6A-37 to ms2i6A-37 is not total.
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Light Absorption Spectroscopy—The bacterial pellets of induced cells as well as the soluble cell free extracts were dark brown. The color survived the heating step (75 °C) during purification. The UV-visible spectrum of the as-isolated recombinant MiaBTm protein was similar to that of the MiaB protein from E. coli (23). It displayed bands at 330, 416, and 460 and a shoulder at 560 nm, a pattern characteristic for [2Fe-2S]+2 clusters (Fig. 6A, and inset a) (23). The iron and sulfur content of the as-isolated protein was low (0.7–1 iron and sulfur/protein). To reconstitute a full iron-sulfur cluster, the apoprotein was prepared by removing iron and sulfur (see "Materials and Methods") and the protein was incubated anaerobically with an excess of ferrous iron and sulfide in the presence of DTT. After desalting on a Sephadex-G25 column, MiaBTm was found to contain approximately 4 iron and 4 sulfur atoms per protein. The UV-visible spectrum of the reconstituted protein is shown in Fig. 6B. It is different from that of the as-isolated protein and more consistent with a [4Fe-4S]+2 cluster (23). Reconstitution of the cluster was also achieved by adding iron and sulfide to the as-isolated form. Its UV-visible spectrum was identical to that shown in Fig. 5B, and, as shown below, its Raman resonance spectrum is very similar to that of the preparation reconstituted from the apo form. During anaerobic reduction of reconstituted MiaBTm with a 10-fold molar excess of sodium dithionite, a bleaching of the solution and a loss of the visible absorption bands were observed.
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EPR Spectroscopy—Both as-isolated and reconstituted proteins were analyzed by EPR spectroscopy. The EPR spectrum of the as-isolated protein is shown in Fig. 7A. It exhibits an isotropic EPR signal centered at g = 2.01 accounting for only 5% of total iron. The relaxation properties and g-tensor of this signal are characteristic for the S = 1/2 ground state of an oxidized [3Fe-4S]+1 cluster. Upon anaerobic reduction with dithionite, the sample gave rise to a new S = 1/2 species, characterized by an axial EPR signal with g values at 2.05 and 1.93, accounting for 40–50% of total iron (Fig. 7B). It became broader at 30 K and could not be detected anymore above 40 K. Such a temperature dependence of the EPR signal and its microwave power saturation properties (data not shown) are characteristic for the S = 1/2 ground state of [4Fe-4S]+1 clusters. Furthermore, the g value of the low field feature is more consistent with a [4Fe-4S]+1 than with a [2Fe-2S]+1 cluster. Non-reduced reconstituted MiaBTm displayed a signal of [4Fe-4S]1+ accounting for as much as 30% of total iron (Fig. 7C). Upon reduction with 2 mM sodium dithionite, this signal increased and accounted for 60% of total iron (Fig. 7D).
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Resonance Raman (RR) Spectroscopy—RR studies of proteins containing [2Fe-2S]+2,+1, [3Fe-4S]+1,0, and [4Fe-4S]+3,+2,+1 clusters in the Fe-S stretching region provide a means of assessing cluster type and redox state (38–41). RR is particularly effective for discriminating between clusters with diamagnetic ground states, such as [4Fe-4S]+2 and [2Fe-2S]+2 clusters, which are not amenable to investigation by EPR or VTMCD spectroscopies, and for providing an initial assessment of the likelihood of partial non-cysteinyl ligation for both [4Fe-4S]+2 (42) and [2Fe-2S]+2 (43–45) clusters.
RR spectra of MiaBTm obtained with 457.9 nm excitation in the Fe-S
stretching region, 200–450 cm–1, are shown
in Fig. 8. In agreement with
the UV-visible absorption spectrum, the resonance Raman spectrum of the
as-isolated MiaBTm (Fig.
8a) is characteristic of a
[2Fe-2S]+2 cluster. The frequency of the lowest energy
mode at 291 cm–1, which corresponds to the
out-of-phase symmetric stretching of the two tetrahedral ligated iron atoms,
is consistent with ligation by four cysteines or three cysteines and one
oxygenic ligand (44,
46). Moreover, the spectrum of
the as-isolated MiaBTm is very similar to those reported for
[2Fe-2S]+2 clusters generated via oxidative degradation
of [4Fe-4S]+2 clusters in other radical-SAM/Fe-S
enzymes, e.g. anaerobic ribonucleotide reductase activating enzyme
(47), pyruvate formate-lyase
activating component (48),
biotin synthase (49), and in
the nitrogenase Fe protein
(50), i.e. broad,
poorly resolved bands centered at 290, 340, and 395
cm–1. In the spectrum of as-isolated MiaBTm, the
trace of [3Fe-4S]+1 clusters that are apparent in the
EPR spectrum are likely to be responsible, at least in part, for the Raman
intensity at 345 cm–1 (see below).
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The resonance Raman spectra of MiaBTm reconstituted anaerobically with iron and sulfide from the apo and as-isolated forms of the enzyme are shown in Fig. 8 (b and c, respectively). Both spectra are very similar and characteristic of [4Fe-4S]+2 clusters (38, 39, 51). Furthermore, because the resonance Raman spectra of [2Fe-2S]+2 and [3Fe-4S]+1 centers are generally 4–6 times more intense than those of [4Fe-4S]+2 centers using 457.9-nm excitation (52, 53), and all the observed bands can be accounted for by a [4Fe-4S]+2 center, the resonance Raman data indicate that [4Fe-4S]+2 clusters are the sole type of oxidized cluster in reconstituted samples. Previous resonance Raman studies of biological [4Fe-4S]+2 centers have shown that the frequency of the most intense band, which corresponds to the symmetric breathing mode of the Fe4S4 cube, provides an indication of cluster ligation (42, 54). The observed frequency of this mode in MiaBTm, 340 cm–1, is at the boundary of the ranges of frequencies reported for [4Fe-4S]+2 clusters with complete cysteinyl ligation (333–339 cm–1) and those having oxygenic ligation at a specific iron site (340–343 cm–1) and hence consistent with but not conclusive concerning the possibility of incomplete cysteinyl ligation.
Additional changes in the resonance Raman spectrum were apparent after reconstituted samples were thawed and exposed to air (Fig. 8d). The bands associated with the [4Fe-4S]+2 were no longer observable, and the resulting spectrum is readily interpretable in terms of overlapping contributions from a [2Fe-2S]+2 cluster analogous to that observed in as-prepared MiaBTm and a [3Fe-4S]+1 similar to those found in bacterial ferredoxins (41, 52). [3Fe-4S]+1 clusters exhibit characteristic resonance Raman spectra with 457.9-nm excitation, which are dominated by an intense band in the range 342–348 cm–1 corresponding to symmetric stretching involving the µ3-S. This band is observed at 346 cm–1 in air-exposed samples of reconstituted MiaBTm. Identical resonance Raman spectra were observed for samples of reconstituted MiaBTm that were exposed to air for 5 and 30 min, indicating that the oxidative cluster degradation occurs rapidly on exposure to air. Confirmation of [3Fe-4S]+1 clusters in air-exposed samples of reconstituted MiaBTm was provided by EPR studies which revealed S = 1/2 resonances analogous to those shown in Fig. 7A corresponding to 25% of the total iron in the sample. Because aerobically isolated MiaBTm contains predominantly [2Fe-2S]+2 clusters with a minor component of [3Fe-4S]+1 clusters accounting for 5% of the total iron (Fig. 7A), we conclude that oxygen-induced degradation of the [4Fe-4S]+2 in anaerobically reconstituted MiaBTm to yield a [2Fe-2S]+2 cluster occurs via a stable [3Fe-4S]+1 cluster intermediate.
VTMCD Spectroscopy—Resonance Raman is of limited utility for assessing cluster type in reduced samples of MiaBTm, because [4Fe-4S]+1 clusters exhibit negligible resonance enhancement with visible excitation (38, 39). Because [4Fe-4S]+1, [3Fe-4S]0, and [2Fe-2S]+1, which are the one-electron-reduced forms of [4Fe-4S]+2, [3Fe-4S]+1 and [2Fe-2S]+2, respectively, are all paramagnetic, VTMCD provides useful supplement to EPR for assessing the nature of the cluster in dithionite-reduced samples via the excited-state properties, as revealed by the complex pattern of temperature-dependent MCD bands in the UV/visible/near-IR region, and the ground-state properties as assessed via variable field and variable temperature MCD saturation magnetization studies (55). The VTMCD spectrum of a dithionite-reduced sample of anaerobically reconstituted MiaBTm is shown in Fig. 9. Saturation magnetization measurements at 540 nm (data not shown) indicate that the MCD bands originate from an S = 1/2 ground state, and hence argue against any significant contribution from S = 3/2 [4Fe-4S]+1 clusters or S = 2 [3Fe-4S]0 clusters. Moreover, the pattern of bands is very similar to that observed for S = 1/2 [4Fe-4S]+1 clusters (55, 56) and quite distinct from those observed for S = 1/2 [2Fe-2S]+1 clusters (40, 55). Parallel EPR studies of the identical sample containing 60% (v/v) ethylene glycol revealed an axial S = 1/2 resonance with g = 2.05 and 1.93, analogous to those shown in Fig. 7 (B–D). All these results are consistent with (i) the reconstituted protein contains [4Fe-4S]+2 and [4Fe-4S]+1 exclusively and (ii) the reduced clusters being exclusively in the [4Fe-4S]+1 state.
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Redox Potential—The redox potential of the reconstituted MiaBTm protein has been measured by square wave voltammetry (Fig. 10). The electrochemical response of the protein was found to be reversible, and the measured redox potential of the [4Fe-4S]+2/+1 was found to be –495 ± 10 mV (versus the normal hydrogen electrode). The value determined here for MiaBTm protein is consistent with redox potential values determined for biotin synthase (57) and the activase of the anaerobic ribonucleotide reductase (58).
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DISCUSSION |
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The results presented in this paper show that the product of the TM0653 gene is a true functional MiaB enzyme, required for the biosynthesis of ms2i6A-37, as indicated by the ability of the corresponding gene to complement the miaB mutation in E. coli. We thus named this protein MiaBTm. The fact that the complementation was effective only at 45 °C is certainly in relation with the thermophilic origin of the MiaBTm protein. We also show that the purified MiaBTm protein is a monomer in solution and that it contains an iron-sulfur cluster. Obviously the MiaBTm protein displays some features already observed with the E. coli enzyme (23). However, when compared with the E. coli enzyme, MiaBTm has several advantages that make this enzyme more suitable for future mechanistic studies. Indeed the E. coli enzyme did not allow the extensive characterization reported here for the first time for a MiaB enzyme. In particular, it proved difficult to get preparations of E. coli MiaB containing homogeneous iron-sulfur cluster composition and spectroscopic data obtained with the E. coli enzyme were hard to interpret. In fact, heterogeneity in cluster composition is currently observed with radical-SAM enzymes. In contrast, clusters in MiaBTm were more stable, as expected for a thermophilic protein, and amenable to thorough spectroscopic characterization which was the objective of the present work. Interestingly, we found conditions for obtaining preparations of MiaBTm containing exclusively either [4Fe-4S] or [2Fe-2S] clusters as clearly shown by resonance Raman spectroscopy.
Under anaerobic conditions, the purified apoprotein could bind up to four atoms of iron and four atoms of sulfur per polypeptide chain, assembled exclusively in a [4Fe-4S] center in both 2+ (oxidized; S = 0) and 1+ (reduced; S = 1/2) states, as shown by UV-visible absorption, resonance Raman and VT-MCD spectroscopy. During reduction with sodium dithionite the proportion of the [4Fe-4S]+1 reduced state increased and no evidence for the presence of reduced [2Fe-2S]+1 or [3Fe-4S]0 clusters could be observed. It is remarkable that the anaerobic reconstitution of the as-isolated form, which contains [2Fe-2S] and [3Fe-4S] clusters, with iron and sulfide also resulted in a preparation containing exclusively [4Fe-4S] clusters.
The spectroscopic studies reported herein show that the [4Fe-4S] cluster of MiaBTm is stable under anaerobic conditions but is degraded by oxygen. This explains why iron and sulfide are lost during purification and why the as-isolated protein contains substoichiometric amounts of iron and sulfur. Aerobically isolated MiaBTm contains mainly diamagnetic [2Fe-2S]+2 clusters, as shown by resonance Raman spectroscopy, together with very small amounts of paramagnetic S = 1/2 [3Fe-4S]+1 clusters, as shown by EPR spectroscopy. Furthermore, during exposure of the reconstituted protein to air, the [4Fe-4S]2+ cluster decomposes generating both [2Fe-2S]+2 and [3Fe-4S]+1 clusters as evidenced by resonance Raman and EPR spectroscopies.
Thus, it appears that the spectroscopic, redox, oxygen sensitivity, and cluster conversion properties of the Fe-S center in MiaB are comparable with those previously reported for other members of the radical-SAM iron-sulfur enzyme family. The presence of essential cysteines within a CysXXXCysXXCys sequence (Cys-150, Cys-154, Cys-157), as already mentioned, together with the requirement of SAM for the conversion of i6A-37 to ms2i6A-37 are further similarities. We thus here confirm that MiaB proteins are members of that enzyme superfamily. Well studied members of this family are lysine 2,3-aminomutase (59), biotin synthase (60), the activating components of ribonucleotide reductase (61), and pyruvate formate-lyase (62). They all contain a [4Fe-4S] cluster chelated by a conserved CysXXXCysXXCys sequence and absolutely require SAM for activity (63).
Oxidative degradation of [4Fe-4S]+2 clusters that constitute the active sites for reductive cleavage of SAM in all radical-SAM Fe-S enzymes generally proceed via [2Fe-2S]+2 cluster (45, 49, 59, 62). The spectroscopic studies presented herein suggest that this cluster transformation occurs via a [3Fe-4S]+1 cluster. In support of this proposal, [3Fe-4S]+1 clusters have been reported in oxygen damaged samples of pyruvate formate-lyase activating enzyme (62), anaerobic ribonucleotide reductase activating enzyme (61), lysine 2,3-aminomutase (59), and lipoic acid synthase (64). However, [3Fe-4S]+1 clusters have yet to be observed in biotin synthase. If oxidative [4Fe-4S]+2 cluster degradation proceeds by the same mechanism in all radical-SAM Fe-S enzymes, we conclude that the [3Fe-4S]+1 intermediate is unstable and occurs only as a transient intermediate in biotin synthase.
The modification of adenosine leading to the thionucleoside ms2i6A at position 37 in most bacterial and eukaryotic tRNAs is an intriguing and unique reaction in many aspects. First, it is very important for normal cell function, as shown from decreased growth rate and cell yield in the case of mutant strains lacking ms2i6A-37 (65). Second, it is still unknown whether MiaB participates in both sulfur insertion and methylation or only to the first process and a second putative enzyme MiaC catalyzes the methylation step. Third, from a chemical point of view, the reaction consists in a highly difficult aromatic C-H to C-S bond conversion. In contrast to the reactions of sulfur insertion into uridine of tRNAs leading to s4U (10) and mnm5s2U (66), which are not redox processes, formation of ms2i6A-37 is an oxidation reaction. However, because MiaB is a member of the radical-SAM enzyme superfamily, it is very likely that its [4Fe-4S] cluster plays a role similar to that of the clusters of other radical-SAM enzymes. We thus speculate that the cluster of MiaB catalyzes a one-electron transfer to SAM for generating a 5'-deoxyadenosyl radical and that the methylthiolation reaction implies such a 5'-deoxyadenosyl radical as the oxidant for a radical activation of the tRNA substrate. Fourth, also in contrast to the reactions leading to s4U (10) and mnm5s2U (66), the sulfur donor during ms2i6A biosynthesis is still unknown and its identification, via genetic studies, is complicated by the fact that sulfur is required both for synthesis of the iron-sulfur cluster and for thiomethylation of adenosine. In the case of the s4U and mnm5s2U, the sulfur donor is likely to be a cysteine persulfide generated by the action of the cysteine desulfurase IscS, because a deletion of the iscS gene in E. coli or Salmonella enterica resulted in non detectable levels of s4U and mnm5s2U (67, 68). In contrast, the same studies showed that IscS was not absolutely required for the biosynthesis of ms2i6A-37 (68).
In the case of biotin synthase, the problem of the sulfur donor has been recently extensively investigated by us and others. These studies have resulted in two conflicting hypothesis. The first one, proposed by J. T. Jarrett, implies a second cluster, a [2Fe-2S] one, as the sulfur donor (69), whereas the second hypothesis implies a persulfide as the donor (70). It is important to note that no evidence for the presence in MiaB of two clusters, one [4Fe-4S] and one [2Fe-2S], could be found, even when cluster reconstitution was achieved from the as-isolated protein. This might suggest that Jarrett's model does not apply to MiaB. We believe that the investigation of MiaB, in particular that from T. maritima, might provide in addition to specific information on formation of ms2i6A-37 new insights into the general question of the mechanism of biological C-H to C-S bond conversion.
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FOOTNOTES |
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¶ To whom correspondence may be addressed: DRDC-CB, Commissariat à l'Energie Atomique-Grenoble, 38054 Grenoble, France. Fax: 33-438789124; E-mail: mfontecave{at}cea.fr.
|| To whom correspondence may be addressed: DRDC-CB, Commissariat à l'Energie Atomique-Grenoble, 38054 Grenoble, France. Fax: 33-438789124; E-mail: mohamed.atta{at}cea.fr.
1 The abbreviations used are: tRNA, transfer ribonucleic acid; ORF, open
reading frame; ms2i6A-37, modified nucleoside
2-methylthio-N-6-isopentenyl adenosine at position 37;
s4U, 4-thiouridine; mnm5s2U,
5-methyaminomethyl-2-thiouridine; RR, resonance Raman; VTMCD, variable
temperature magnetic circular dichroism; MCD, magnetic circular dichroism;
SAM, S-adenosylmethionine; Fe-S, iron-sulfur cluster; DTT,
dithiothreitol; W, watt(s); LB, Luria Bertani; HPLC, high performance liquid
chromatography; T, tesla.
2 F. Pierrel, G. R. Björk, M. Fontecave, and M. Atta, unpublished
results.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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