Structure Determination by Combination of CW and Pulsed '2-D' Orientation-Selective ^1,2H Q-Band Electron-Nuclear Double Resonance: Identification of the Protonated Oxygenic Ligands of Ribonucleotide Reductase Intermediate X

The structure of a paramagnetic center can be determined by analysis of the electron-nuclear hyperfine and nuclear-quadrupole coupling tensors of interacting nuclei. These tensors can be determined for a frozen-solution sample by analyzing the orientation-selective 2-D pattern generated by collecting ENDOR spectra at multiple fields across the entire EPR envelope. This process requires careful comparison of experimental and simulated ENDOR patterns, which requires that the spectra must be collected in such a manner as to give reliable peak intensities and shapes as well as frequencies. This requirement usually cannot be met by either CW or pulsed-ENDOR protocols alone, but the goal can be reached by a combination of CW and pulsed-ENDOR measurements. This process is illustrated by showing how continuous wave (CW) and pulsed Q-Band ^1,2H ENDOR measurements have been used to identify the types of protonated oxygen (OH_x) species coordinated to diiron center of Intermediate X formed during oxygen activation by the R2 subunit of Escherichia coli Ribonucleotide Reductase (RNR), as well as their disposition relative to the ferric and ferryl iron ions: X contains the [(H_xO)Fe^IIIOFe^IV] fragment.