The tyrosyl free radical of recombinant ribonucleotide reductase from Mycobacterium tuberculosis is located in a rigid hydrophobic pocket.

The tyrosyl free radical in protein R2-2 of class Ib ribonucleotide reductase (RNR) fromMycobacterium tuberculosis is essential for the enzymatic activity and has an EPR spectrum remarkably similar to that of the tyrosyl radical YD* in PSII. The EPR relaxation properties of the radical suggest a very weak exchange coupling between the two redox centers, the radical and the diferric cluster. The tyrosyl radical gives almost identical EPR spectra in the temperature interval 10-293 K. We conclude that the tyrosyl radical sits in a rigid pocket. Two ring protons and one beta-methylene proton account for the major anisotropic hyperfine interactions. A high-frequency EPR spectrum of the radical showed a resolved gx = 2. 0092, indicating that a hydrogen bond to the phenolic oxygen of the radical is absent. Theoretical modeling studies based on the structural data known for Salmonella typhimurium class Ib RNR protein R2F revealed a hydrophobic wall aligned with the radical harboring residue Y110. The distance between the phenolic oxygen of the radical and the diferric cluster is longer in the two class Ib nrdF R2 proteins than in other characterized class Ia R2 proteins. The tyrosyl radical in protein R2-2 from M. tuberculosis was accessible to direct reduction by dithionite in the absence of a mediator. The radical could be partly regenerated when the system was exposed to O2 after the completion of anaerobic reduction. This indicates that the Fe3+ ions also had become reduced by dithionite.