title: EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein.
abstract: Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (gamma-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g(av) < 2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct mu-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g(av) < 1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a mu-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.
authors.affiliation: Department of Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Davydov
authors.forename: A
authors.initials: A
authors.lastname: Liu
authors.forename: A
authors.initials: A
authors.lastname: Gräslund
mesh_terms: D004227:Dithionite / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005720:Gamma Rays; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology*; D019159:Nonheme Iron Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry*
chemical_list: D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; D004227:Dithionite; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit