biostudies-literatureUnknownWestmoreland DENIGMS NIH HHSRibonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic-electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N₃CDP) to trap a wild-type α2β2 complex of <i>E. coli</i> class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a mid-turnover state. Instead of N₃CDP in the active site, forward RT has resulted in N₂ loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. To the best of our knowledge, this is the first time an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a pre-turnover PCET pathway and suggesting how PCET is gated at the α-β interface. This N₃CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.bioRxiv : the preprint server for biology2024.10.09.617422https://www.ebi.ac.uk/biostudies/studies/S-EPMC11482829biostudies-literature2.6-A resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N&lt;sub&gt;3&lt;/sub&gt;CDP.PMC11482829R01 GM029595R35 GM126982F32 GM145072R01 GM047274Nocera DGKim AKang GWestmoreland DEStubbe JFeliciano PRDrennan CLCui Cfalse2.6-A resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N&lt;sub&gt;3&lt;/sub&gt;CDP.Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic-electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N₃CDP) to trap a wild-type α2β2 complex of <i>E. coli</i> class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a mid-turnover state. Instead of N₃CDP in the active site, forward RT has resulted in N₂ loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. To the best of our knowledge, this is the first time an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a pre-turnover PCET pathway and suggesting how PCET is gated at the α-β interface. This N₃CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.2024-01-01T00:00:00Z2024 Oct2025-04-18T13:12:55.23Z2025-04-06T22:42:49.282ZS-EPMC114828293941610310.1101/2024.10.09.617422