Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.

Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a proteinbound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.T he cellular translation machinery contains essential components such as tRNAs. To achieve their function, they feature a great variety of well-conserved posttranscriptional chemical modifications. Sulfur is present in several of these modified nucleosides: thiouridine and derivatives (s 4 U8, s 2 U34, and m 5 s 2 U54), 2-thioadenosine derivatives (ms 2 i 6 A37 and ms 2 t 6 A37), and 2-thiocytidine (s 2 C32). However, mechanisms of sulfur insertion into tRNAs are largely unknown, and the enzymes responsible for these reactions are incompletely characterized. Whereas redox conversion of a C-H to a C-S bond (synthesis of ms 2 i 6 A37 and ms 2 t 6 A37) depends on redox enzymes from the Radical-S-adenosyl-L-methionine iron-sulfur enzyme family, simple nonredox conversion of C = O to C = S group (synthesis of s 2 U34 and s 4 U8) is not expected to require such redox clusters. Intriguingly, we recently discovered that the ATPdependent formation of s 2 C32 in some tRNAs is catalyzed by an iron-sulfur enzyme, TtcA (1). However, the role of its cluster has not been defined. In the same superfamily, TtuA enzymes catalyze the C2-thiolation of uridine 54 in the T loop of thermophilic tRNAs (Fig. 1A), allowing stabilization of tRNAs at high temperature in thermophilic microorganisms. Sequences analysis shows that they share conserved cysteines and ATP binding motif (Fig. S1). Here, we report a detailed biochemical and structural characterization of TtuA that shows the presence of a [4Fe-4S] cluster essential for activity. The crystal structures of Pyrococcus horikoshii TtuA (PhTtuA) show that the cluster, chelated by only three cysteines, is adjacent to the ATP binding site. The presence of electron density near the fourth iron, nonbonded to the protein, indicates that the cluster can bind an exogenous substrate. We propose that thiolation occurs via sulfur binding to the cluster and transfer to the tRNA substrate. The fact that the catalytic [4Fe-4S] cluster serves as a sulfur carrier during a nonredox ...

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