Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase

Matthias Kolberg, G. Bleifuss, B. M. Sjöberg, A. Gräslund, W. Lubitz, F. Lendzian, G. Lassmann

Research output: Contribution to journalArticle

17 Citations (Scopus)

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 CeIV/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 CeIV/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 β-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.

Original languageEnglish (US)
Pages (from-to)57-68
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume397
Issue number1
DOIs
StatePublished - Jan 1 2002

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Spin Trapping
Ribonucleotide Reductases
Photolysis
Electron Spin Resonance Spectroscopy
Escherichia coli
Tensors
Cysteine
Paramagnetic resonance
Lasers
Reaction products
Protons
Reactive Oxygen Species
Nitric Oxide
Proteins
Oxidation
Temperature
Substrates

Keywords

  • EPR
  • Nitroso-thiol photolysis
  • Protein-based thiyl radicals
  • Ribonucleotide reductase
  • Spin trapping
  • Sulfinyl radicals
  • Thiol oxidation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase. / Kolberg, Matthias; Bleifuss, G.; Sjöberg, B. M.; Gräslund, A.; Lubitz, W.; Lendzian, F.; Lassmann, G.

In: Archives of Biochemistry and Biophysics, Vol. 397, No. 1, 01.01.2002, p. 57-68.

Research output: Contribution to journalArticle

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