Quick and Spontaneous Transformation between [3Fe-4S] and [4Fe-4S] Iron-Sulfur Clusters in the tRNA-Thiolation Enzyme TtuA.

Iron-sulfur (Fe-S) clusters are essential cofactors for enzyme activity. These Fe-S clusters are present in structurally diverse forms, including [4Fe-4S] and [3Fe-4S]. Type-identification of the Fe-S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe-4S] and [3Fe-4S] clusters in particular is challenging because of their rapid transformation in response to oxidation-reduction events. In this study, we focused on the relationship between the Fe-S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe-4S]-TtuA, prepared [3Fe-4S]-TtuA by oxidizing [4Fe-4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe-S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe-4S]-TtuA spontaneously transforms into [4Fe-4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe-4S]-TtuA was inactive [3Fe-4S]-TtuA, its activity recovered to a significant level compared to [4Fe-4S]-TtuA after one hour, corresponding to an increase of [4Fe-4S]-TtuA in the solution. Our findings reveal that [3Fe-4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe-S cluster type used by the tRNA-thiolation enzyme.

The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus.

TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed.

Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA.

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

Crystallographic study of the 2-thioribothymidine-synthetic complex TtuA-TtuB from Thermus thermophilus.

The ubiquitin-like protein TtuB is a sulfur carrier for the biosynthesis of 2-thioribothymidine (s2T) at position 54 in some thermophilic bacterial tRNAs. TtuB captures a S atom at its C-terminus as a thiocarboxylate and transfers it to tRNA by the transferase activity of TtuA. TtuB also functions to suppress s2T formation by forming a covalent bond with TtuA. To explore how TtuB interacts with TtuA and switches between these two different functions, high-resolution structure analysis of the TtuA-TtuB complex is required. In this study, the TtuA-TtuB complex from Thermus thermophilus was expressed, purified and crystallized. To mimic the thiocarboxylated TtuB, the C-terminal Gly residue was replaced with Cys (G65C) to obtain crystals of the TtuA-TtuB complex. A Zn-MAD data set was collected to a resolution of 2.5 Å. MAD analysis successfully determined eight Zn sites, and a partial structure model composed of four TtuA-TtuB complexes in the asymmetric unit was constructed.