PMID:10430881
| Citation |
Lawrence, CC, Bennati, M, Obias, HV, Bar, G, Griffin, RG and Stubbe, J (1999) 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. Proc. Natl. Acad. Sci. U.S.A. 96:8979-84 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Amino Acid Substitution; Anions; Binding Sites; Cytidine Diphosphate/chemistry; Cytidine Diphosphate/metabolism; Disulfides; Electron Spin Resonance Spectroscopy/methods; Escherichia coli/enzymology; Free Radicals; Mutagenesis, Site-Directed; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Ribonucleotide Reductases/chemistry; Ribonucleotide Reductases/metabolism; Spectrophotometry; Tyrosine |
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