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 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.
Lawrence
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CC
Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Bennati
M
M
Obias
H V
HV
Bar
G
G
Griffin
R G
RG
Stubbe
J
J
D019943:Amino Acid Substitution; D000838:Anions; D001665:Binding Sites; D003565:Cytidine Diphosphate / Q000737:chemistry* / Q000378:metabolism*; D004220:Disulfides; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D013053:Spectrophotometry; D014443:Tyrosine
D000838:Anions; D004220:Disulfides; D005609:Free Radicals; D011994:Recombinant Proteins; D014443:Tyrosine; D003565:Cytidine Diphosphate; D012264:Ribonucleotide Reductases