Ribonucleotide reductase (RNR) is a radical enzyme that catalyzes de novo biosynthesis of deoxyribonucleotides. The catalytically required tyrosyl free radical in class I RNRs is produced in conjunction with a mu-oxo-bridged diferric center in protein R2, 35 Angstrom away from the substrate-binding site in protein R1. High-field EPR at 285 GHz was applied to probe the environment of the tyrosyl radical in both native and reconstituted samples of protein R2-2 of class To RNR from Mycobacterium tuberculosis. Two distinct peaks (2.0080, 2.0092) of the g(x) component were observed in the spectrum from freshly purified native R2-2 protein as well as from ferrous iron/oxygen reconstituted apoprotein R2-2. The g(x)= 2.0092 peak was relatively stable, whereas the g(x) = 2.0080 peak decayed after freezing-thawing and storage. The two peaks corresponding to total g-anisotropies of 0.007 and 0.006, respectively, are interpreted in terms of a non-H-bonded and a weakly H-bonded fraction of the radicals. The hydrogen bond may be provided from a well-ordered water molecule in the vicinity of the radical-iron site, based on a model study of the protein. This first direct observation of conformational heterogeneity of the local tyrosyl radical environment may be important for understanding the role of the radical in the proposed long-range radical transfer through a chain of H-bonded residues from the radical site in protein R2-2 to the catalytic site in protein R1. The enzyme may become activated by connection of a complete H-bonded chain to the radical.