Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleoticle reduction occurs, and R2 contains the diiron tyrosyl radical (Y center dot) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N3UDP), have provided much insight into the reduction mechanism. N3UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y center dot in R2 and formation of a nitrogen-centered radical (N center dot) covalently attached to C225 (R-S-N center dot-X) in the active site of R1. N-2 is lost prior to No formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with No, it was proposed that No is also covalently attached to the nucleoticle through either the oxygen of the T-OH (R-S-N center dot-O-R') or the T-C (R-S-N center dot-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[O-17]-N3UDP and No was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the No signal was detected and the spectrum simulated to obtain the [O-17] hyperfine tensor. DIFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are most consistent with the R-S-N center dot-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.