Tryptophan and Tyrosine Radicals in Ribonucleotide Reductase:  A Comparative High-Field EPR Study at 94 GHz

• Published in: Biochemistry (Easton) 2001-12, Vol.40 (50), p.15362-15368
• Main Authors: Bleifuss, Günther, Kolberg, Matthias, Pötsch, Stephan, Hofbauer, Wulf, Bittl, Robert, Lubitz, Wolfgang, Gräslund, Astrid, Lassmann, Günter, Lendzian, Friedhelm
• Format: Article
• Language: English
• Subjects: Animals; Electron Spin Resonance Spectroscopy; Escherichia coli - enzymology; Escherichia coli - genetics; Free Radicals - chemistry; Mice; Mutagenesis, Site-Directed; Protein Subunits; Ribonucleotide Reductases - chemistry; Ribonucleotide Reductases - genetics; Tryptophan - chemistry; Tyrosine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010707d

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe2+ and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.