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) (By similarity). This modification allows thermal stabilization of tRNAs in thermophilic microorganisms, and is required for cell growth at high temperatures (By similarity). 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 M.jannaschii. 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.