EPR evidence of two structurally different diferric sites in Mycobacteriumtuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyro syl free radical) and met (without radical) forms of protein R2-2 in a clas s Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temper ature reduction (gamma-irradiation) at 77 K. The primary mixed-valent EPR s ignal is a mixture of two components with axial symmetry and g(a nu) < 2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetical ly coupled high spin ferric/ferrous sites. The two components in the primar y EPR signal can be explained by the existence of two structurally distinct mu-oxo-bridged diferric centers, possibly related to structural heterogene ity around the iron site, and/or different properties of the two polypeptid e chains in the homodimeric protein after the radical reconstitution reacti on. Annealing of the irradiated R2-2 samples to 143 K transforms the primar y EPR signal into a rhombic spectrum characterized by g(a nu) < 1.8 and obs ervable only below 25 K. This spectrum is assigned to a partially relaxed f orm with a mu-hydroxo-bridge. Further annealing at 228 K produces a new com plex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction o f Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite. (C) 2000 Else vier Science S.A. All rights reserved.