Orientation of the tyrosyl radical in salmonella typhimurium class Ib ribonucleotide reductase determined by high-field EPR of R2F single crystals. [J Biol Chem] Journal article

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of S. typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from E. coli. No structural information has been available for the active tyrosyl radical in R2F. Here we report on high-field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Y105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Y105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Y105-OH from the X-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands towards similar values as observed earlier for Y122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Y105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining X-ray and high-field-EPR crystallography.

J Biol Chem