New paramagnetic species formed at the expense of the transient tyrosyl radical in mutant protein R2 F208Y of Escherichia coli ribonucleotide reductase.
The highly conserved residue F208 in protein R2 of E. coli ribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122. in wild type R2. Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g = 2.003) formed concomitantly with decay of the transient tyrosyl radical Y122. (g = 2.005). This new paramagnetic species (denoted Z) was stable for weeks at 4 degrees C and visible by EPR only below 50 K. The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species. The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2. An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F. In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122. and the new species Z increased considerably in the reconstitution reaction. The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical.
Liu
A
A
Department of Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.
Sahlin
M
M
Pötsch
S
S
Sjöberg
B M
BM
Gräslund
A
A
D001665:Binding Sites; D004295:Dihydroxyphenylalanine; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D008956:Models, Chemical; D009154:Mutation; D019159:Nonheme Iron Proteins / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D005609:Free Radicals; D019159:Nonheme Iron Proteins; D014443:Tyrosine; D010649:Phenylalanine; D004295:Dihydroxyphenylalanine; D012264:Ribonucleotide Reductases