PMID:11747310
| Citation |
Kolberg, M, Bleifuss, G, Sjöberg, BM, Gräslund, A, Lubitz, W, Lendzian, F and Lassmann, G (2002) Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase. Arch. Biochem. Biophys. 397:57-68 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1006/abbi.2001.2658 |
| Keywords |
Electron Spin Resonance Spectroscopy/methods; Escherichia coli/enzymology; Lasers; Models, Chemical; Nitric Oxide/chemistry; Oxygen/metabolism; Photolysis; Protein Binding; Ribonucleotide Reductases/chemistry; Spin Labels; Spin Trapping/methods |
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