Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor and R1 is the site of the conversion of nucleoside diphosphates to 2-deoxynucleoside diphosphates. A mutant in the R1 subunit of Escherichia coli RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process. Cytidine 5-diphosphate was incubated with E441Q RNR, and the reaction was monitored by using stopped-f low UV-vis spectroscopy and highfrequency (140 GHz) time-domain EPR spectroscopy. These studies revealed loss of the Y⅐ and formation of a disulfide radical anion and present experimental mechanistic insight into the reductive half-reaction catalyzed by RNR. These results support the proposal that the protonated E441 is required for reduction of a 3-ketodeoxynucleotide by a disulfide radical anion. On the minute time scale, a second radical species was also detected by high-frequency EPR. Its g values suggest that this species may be a 4-ketyl radical and is not on the normal reduction pathway. These experiments demonstrate that high-field time-domain EPR spectroscopy is a powerful new tool for deconvolution of a mixture of radical species.Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms (1-3). These enzymes have been divided into four classes based on the metallo-cofactor required to initiate the radical-dependent nucleotide-reduction process. Recent structural studies on class I and class III RNRs (4-6) support the original proposal of Stubbe and coworkers that, despite the differences in the metallo-cofactors in the various classes of RNRs, the function of each cofactor is to generate a thiyl radical that, via a common mechanism, initiates nucleotide reduction by 3Ј-hydrogen atom abstraction (Scheme 1) (7-9). A further commonality between class I and II RNRs is the presence of a conserved glutamate residue adjacent to the two cysteine residues that deliver the reducing equivalents essential for the reduction process (5). This paper provides evidence that the role of this glutamate (in the protonated form) is to facilitate the reduction of the 3Ј-ketodeoxynucleotide 3 (Scheme 1) by a disulfide radical anion intermediate.Much is known about the initial stages of the reduction process (1, 10). There is direct evidence that in the class II, adenosylcobalamin-dependent RNRs, the metallo-cofactor generates a thiyl radical in a kinetically-competent fashion (11,12). Recent studies with a class I RNR containing a diferric tyrosyl radical (Y⅐) cofactor and a class II RNR using cytidine nucleotide analogs provided direct evidence that the thiyl radical generates a 3Ј-nucleotide analog radical (13-16). Once 1 is generated, there is also excellent model precedent that a ketyl radical 2 is generated subsequent to loss of water from the 2Ј position of the nucleotide (17, 18).The mechanism for the reduction of 2 has received less experimental a...

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 nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss 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. N(2) is lost prior to N* 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 N*, it was proposed that N* is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N*-O-R') or the 3'-C (R-S-N*-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[(17)O]-N(3)UDP and N* was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N* signal was detected and the spectrum simulated to obtain the [(17)O] hyperfine tensor. DFT 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*-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.