High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5 '-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 a nd R2. The R2 subunit contains the essential diferric cluster-tyrosyl radic al CY) cofactor and R1 is the site of the conversion of nucleoside diphosph ates to 2'-deoxynucleoside diphosphates. A mutant in the R1 subunit of Esch erichia coli RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process. Cytidine 5'-diphosphate was in cubated with E441Q RNR, and the reaction was monitored by using stopped-flo w UV-vis spectroscopy and high-frequency (140 GHz) time-domain EPR spectros copy. These studies revealed loss of the Y-. and formation of a disulfide r adical anion and present experimental mechanistic insight into the reductiv e half-reaction catalyzed by RNR. These results support the proposal that t he protonated E441 is required for reduction of a 3'-ketodeoxynucleotide by a disulfide radical anion. On the minute time scale, a second radical spec ies 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 pathw ay. These experiments demonstrate that high-field time-domain EPR spectrosc opy is a powerful new tool for deconvolution of a mixture of radical specie s.