Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase. |
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Authors: | Matthias Kolberg Günther Bleifuss Britt-Marie Sj?berg Astrid Gr?slund Wolfgang Lubitz Friedhelm Lendzian Günter Lassmann |
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Affiliation: | Max-Volmer-Laboratorium für Biophysikalische Chemie, Institut für Chemie, Technische Universit?t Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany. |
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Abstract: | In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used. |
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Keywords: | protein-based thiyl radicals sulfinyl radicals thiol oxidation nitroso-thiol photolysis ribonucleotide reductase EPR spin trapping |
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