During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.crystallography ͉ metalloenzyme ͉ ribozyme ͉ splicing G roup I intron splicing proceeds through two consecutive transesterification reactions separated by a conformational change. The first step proceeds by the addition of an exogenous G (␣G) to the 5Ј end of the intron. This covalently releases the 5Ј exon, but the exon remains bound through base-pairing and tertiary hydrogen bonds (1, 2). A conformational change moves the ␣G out of the active site and exposes a portion of the internal guide sequence (IGS) that is complementary to the 3Ј exon (3). The IGS then base-pairs to both the 5Ј and 3Ј exons, orienting them for the second step of splicing (reaction state pre-2S). During exon ligation the 5Ј exon attacks the scissile phosphate between the last nucleotide of the intron (⍀G) and the 3Ј exon (reaction state post-2S). After ligation, the exons are released from the intron in at least two steps (4, 5). The tertiary contacts to the exon-IGS helix (P1-P10 or substrate helix) are disrupted, resulting in an open or undocked complex (6, 7), and then the substrate helix dissociates to release the ligated exons (8, 9).Recent crystal structures have provided a molecular view of a subset of the covalent and conformational states along the splicing pathway. Two structures of the Azoarcus sp. BH72 pre-tRNA Ile group I intron captured the intron bound to both exons before the second step of splicing (pre-2S) (10, 11). In the first structure (deoxy pre-2S), the ligation reaction was prevented by the inclusion of 2Ј-deoxyribose substitutions at ⍀G, an essential hydroxyl group, and at three other positions (10). The second structure (ribo pre-2S) contained a ribose at all positions, including ⍀G, except U-1 (the last nucleotide of the 5Ј exon) (11). The resulting complex had some activity in solution and within the crystals. Two other group I intron structures were reported soon after the Azoarcus intron. The firs...