Catalyzes the ATP-dependent 2-thiolation of 5-methyluridine residue at position 54 in the T loop of tRNAs, leading to 5-methyl-2-thiouridine (m(5)s(2)U or s(2)T) (PubMed:28655838). This modification allows thermal stabilization of tRNAs in thermophilic microorganisms, and is required for cell growth at high temperatures (By similarity). Can use free sulfide as sulfur source in vitro, which may be also the sulfur source in vivo (PubMed:28655838). 5-methyluridine(54) in tRNA + ATP + hydrogen sulfide = 5-methyl-2-thiouridine(54) in tRNA + AMP + diphosphate Binds 1 [4Fe-4S] cluster per subunit. The cluster is chelated by three Cys residues, the fourth Fe with a free coordination site may bind a small ligand, such as exogenous sulfide, thus acting as a sulfur carrier. tRNA modification. Homodimer. In TtuA from T.thermophilus, the sulfur inserted into the nucleoside comes from the C-terminal thiocarboxylate of TtuB, but there is no TtuB ortholog in P.horikoshii. Free sulfide has been shown to be present at relatively high concentrations within thermophilic archaea, and may be the sulfur source in vivo. The thiolation reaction likely consists of two steps: a first activation step by ATP to form an adenylated intermediate of the target base of tRNA, and a second nucleophilic substitution step of the sulfur (S) atom supplied by the hydrosulfide attached to the Fe-S cluster. Belongs to the TtcA family. TtuA subfamily.