Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-Å x-ray data

Martin Högbom, Marcus Galander, Martin Andersson, Matthias Kolberg, Wulf Hofbauer, Günter Lassmann, Pär Nordlund, Friedhelm Lendzian

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Abstract

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122°). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-Å x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122° obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography.

Original languageEnglish (US)
Pages (from-to)3209-3214
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number6
DOIs
StatePublished - Mar 18 2003

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Ribonucleotide Reductases
X-Rays
Proteins
Ribonucleotides
Crystallography
Tyrosine
Binding Sites
Escherichia coli
DNA

ASJC Scopus subject areas

  • General

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Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-Å x-ray data. / Högbom, Martin; Galander, Marcus; Andersson, Martin; Kolberg, Matthias; Hofbauer, Wulf; Lassmann, Günter; Nordlund, Pär; Lendzian, Friedhelm.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, No. 6, 18.03.2003, p. 3209-3214.

Research output: Contribution to journalArticle

Högbom, Martin ; Galander, Marcus ; Andersson, Martin ; Kolberg, Matthias ; Hofbauer, Wulf ; Lassmann, Günter ; Nordlund, Pär ; Lendzian, Friedhelm. / Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-Å x-ray data. In: Proceedings of the National Academy of Sciences of the United States of America. 2003 ; Vol. 100, No. 6. pp. 3209-3214.
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abstract = "The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122°). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-{\AA} x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122° obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography.",
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