| Abstract
| - Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotidesproviding the monomeric precursors required for DNA replication and repair. The class I RNRs are composedof two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, andR2 contains the diiron tyrosyl radical (Y•) cofactor essential for radical initiation on R1. Mechanism-basedinhibitors, such as 2‘-azido-2‘-deoxyuridine-5‘-diphosphate (N3UDP), have provided much insight into thereduction mechanism. N3UDP is a stoichiometric inactivator that, upon interaction with RNR, results inloss of the Y• in R2 and formation of a nitrogen-centered radical (N•) covalently attached to C225 (R−S−N•−X) in the active site of R1. N2 is lost prior to N• formation, and after its formation, stoichiometric amountsof 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfineinteractions associated with N•, it was proposed that N• is also covalently attached to the nucleotide througheither the oxygen of the 3‘-OH (R−S−N•−O−R‘) or the 3‘-C (R−S−N•−C−OH). To distinguish betweenthe proposed structures, the inactivation was carried out with 3‘-[17O]-N3UDP and N• was examined by 9and 140 GHz EPR spectroscopy. Broadening of the N• signal was detected and the spectrum simulatedto obtain the [17O] hyperfine tensor. DFT calculations were employed to determine which structures are inbest agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are mostconsistent with the R−S−N•−C−OH structure and provide evidence for the trapping of a 3‘-ketonucleotidein the reduction process.
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