High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: The tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast

High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y .)-diiron cofactor in the Y2-containing R2 subunit of ri bonucleotide reductase (RNR) from yeast. The present work represents the fi rst use of 140-GHz time domain EPR and ENDOR to examine this system and dem onstrates the capabilities of the method to elucidate the electronic struct ure and the chemical environment of protein radicals. Low-temperature spin- echo-detected EPR spectra of yeast Y . reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y . from E. coli RNR, a substantial upfield shift of the gl-value is observed. The o rigin of the shift in g(1) was investigated by 140-GHz H-1 and H-2 pulsed E NDOR experiments of the Y2-containing subunit in protonated and D2O-exchang ed buffer. H-2 ENDOR spectra and simulations provide unambiguous evidence f or one strongly coupled H-2 arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule. Orientation -selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bon d with respect to the molecular symmetry axes and the bond length (1.81 Ang strom). Finally, we have performed saturation recovery experiments and obse rved enhanced spin lattice relaxation rates of the Y . above 10 K. Ar tempe ratures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction bet ween Y . and the first excited paramagnetic state of the diiron cluster adj acent to it. From the activation energy of the rates, we determine the exch ange interaction between the two irons of the cluster, J(exc) = -85 cm(-1). The relaxation mechanism and the presence of the hydrogen bond are discuss ed in terms of the differences in the structure of the Y . -diiron cofactor in yeast Y2 and other class I R2s.