标题：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
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：16
页码：8979-8984
DOI：10.1073/pnas.96.16.8979
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y*) 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-flow UV-vis spectroscopy and high-frequency (140 GHz) time-domain EPR spectroscopy. These studies revealed loss of the Y* 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.
