A large number of post-translational modifications are found in anticodon stem-loop (ASL) of tRNAs, prominently located at the wobble position. Queuosine (Q) is a representative hypermodified nucleoside in the ASL encoding Asn, Asp, His or Tyr. Epoxyqueuosine reductase QueH is responsible for the final step of the biosynthetic pathway to Q, catalyzing a reduction of epoxyqueuosine. The structure of QueH from <i>T. maritima</i> showed that the enzyme contains two metal binding sites: an iron-sulfur cluster coordinated with a four-cysteines, and an unpredicted single metal binding site formed two cysteines and an aspartate ligand. To understand what the single metal is and how QueH catalyze the deoxygenation of epoxyqueuosine, we mutated one of the two cysteines and determined the structure of the mutant using X-ray crystallography. Our structure shows that the two cysteines are indispensable for the single metal binding site. Chemical composition analysis and inductively coupled plasma mass spectrometry (ICP-MS) analysis were employed confirming the [4Fe-4S] cluster along with a single metal zinc. Electron paramagnetic resonance (EPR) was also used to characterize the [4Fe-4S] cluster and demonstrate the redox properties. The information in these studies provides insight into the mechanism of the final step of the Q biosynthetic pathway catalyzed unique enzymology.

Structural and functional studies of epoxyqueuosine reductase QueH in the biosynthesis of queuosine modified tRNA

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