The tRNA wobble position modifications 5-carboxymethoxyuridine (cmo5U) and 5-methoxycarbonylmethoxyuridine (mcmo5U) are essential for expanding the decoding capacity of tRNAs in many bacterial species.1 These modifications are initiated by hydroxylation of the C5 position of the uracil base. TrhP, tRNA hydroxylation protein, catalyzes this critical modification in E. coli in the absence of molecular oxygen. TrhP contains four highly conserved cysteine residues (CX6CX15CX3C) that are necessary for its hydroxylation activity in vivo.1 Although previous studies have suggested that TrhP harbors an iron-sulfur cluster, the cluster's nuclearity and electronic structure have not been thoroughly investigated. In this study, we employ UV-vis and electron paramagnetic resonance (EPR) spectroscopy to investigate the type, coordination environment, and redox properties of the iron-sulfur cluster within TrhP. Our results demonstrate that TrhP can accommodate different types of iron-sulfur clusters depending on the preparation conditions. Notably, the spectroscopic data suggest the presence of a labile [4Fe-4S] cluster, similar to site-differentiated clusters found in other well-characterized enzymes. These findings offer a valuable foundation for understanding the biochemical mechanism behind the oxygen-independent hydroxylation reaction facilitated by TrhP.