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
| Autor: | Honorio V. Obias, Robert G. Griffin, Galit Bar, JoAnne Stubbe, Christopher C. Lawrence, Marina Bennati |
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| Rok vydání: | 1999 |
| Předmět: |
Anions
Ribonucleotide Free Radicals Stereochemistry Protein subunit Photochemistry Cofactor Cytidine Diphosphate law.invention chemistry.chemical_compound law Ribonucleotide Reductases Escherichia coli Disulfides Electron paramagnetic resonance Cytidine diphosphate Multidisciplinary Binding Sites biology Electron Spin Resonance Spectroscopy Cytidine Biological Sciences Recombinant Proteins Ribonucleotide reductase chemistry Amino Acid Substitution Spectrophotometry biology.protein Mutagenesis Site-Directed Tyrosine Nucleoside |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America. 96(16) |
| ISSN: | 0027-8424 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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