The mitochondrial tyrosyl tRNA synthetases (mtTyrRS) from certain fungii are found to be bifunctional enzymes that aid in group I intron splicing in addition to charging tRNA[superscript Tyr]. This splicing activity is conferred by several insertions that are unique to these mtTyrRS. Initial biochemical evidence suggested the similar tertiary structures of the tRNA and the intron enable binding of the protein to both. However, a recently solved co-crystal structure showed that the tRNA and intron were bound on opposite faces of the protein. The intron was bound almost exclusively by a novel surface formed by several insertions in the protein. This work presents the structure of the C-terminal domain of the A. nidulans mtTyrRS (PDB ID -- 2ktl). NMR results show that the C-terminal domain contains an S4 fold with a mixed [beta]-sheet and two anti-parallel [alpha]-helices that pack against these strands. The strands [beta]1 and [beta]5 are parallel, and [beta]2 to [beta]5 are arranged anti-parallel to each other. The C-terminal domain from A. nidulans mtTyrRS has three insertions in its sequence that make it almost twice the size of bacterial TyrRS. NMR results show that insertion 3 at the N-terminus of the domain is flexible. Insertion 4 is contained in the loop connecting [beta]2-[beta]3 and does not have a well defined structure. Insertion 5 and the C-terminal extension form two helices, [alpha]5 and [alpha]6 that fold away from the core of the protein. An extended helix ([alpha]4) between strands [beta]3 and [beta]4 was identified by NMR. Based on structural alignments with bacterial TyrRS, this helix was classified as a novel insertion 4b in the C-terminal domain. Conserved positively charged residues used to bind the tRNA are found in the turn between the anti-parallel [alpha]-helices and the turn connecting strands [beta]4-[beta]5. Based on a comparison with other TyrRS structures, the three insertions are positioned away from the tRNA binding site. The insertions form a novel RNA binding surface that could interact with the intron. Since these insertions are found in loop and termini regions, they could be a structural adaptation acquired by these splicing mtTyrRS. NMR spectra of the full length TyrRS from B. stearothermophilus and mtTyrRS from A. nidulans indicate that the motion of the C-terminal domain is coupled to that of the full length protein. This provides new information regarding the organization of the full length TyrRS. / text