Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.
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.
Kolberg
Matthias
M
Max-Volmer-Laboratorium für Biophysikalische Chemie, Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany.
Bleifuss
Günther
G
Sjöberg
Britt-Marie
BM
Gräslund
Astrid
A
Lubitz
Wolfgang
W
Lendzian
Friedhelm
F
Lassmann
Günter
G
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*
D013113:Spin Labels; D009569:Nitric Oxide; D012264:Ribonucleotide Reductases; D010100:Oxygen