title: Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.
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.
authors.affiliation: Max-Volmer-Laboratorium für Biophysikalische Chemie, Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kolberg
authors.forename: Günther
authors.initials: G
authors.lastname: Bleifuss
authors.forename: Britt-Marie
authors.initials: BM
authors.lastname: Sjöberg
authors.forename: Astrid
authors.initials: A
authors.lastname: Gräslund
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lubitz
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Lendzian
authors.forename: Günter
authors.initials: G
authors.lastname: Lassmann
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D007834:Lasers; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013113:Spin Labels; D019097:Spin Trapping / Q000379:methods*
chemical_list: D013113:Spin Labels; D009569:Nitric Oxide; D012264:Ribonucleotide Reductases; D010100:Oxygen