Identification of protonated oxygenic ligands of ribonucleotide reductase intermediate X

We previously used a combination of continuous-wave (CW) and pulsed electron-nuclear double resonance (ENDOR) protocols to Identify the types of protonated oxygen (OH_x) species and their disposition within the Fe^III/Fe^IV cluster of intermediate X, the direct precursor of the essential diferric-tyrosyl radical cofactor of the β2 subunit of Escherichia coli ribonucleotide reductase (RNR). We concluded that X contains the [(H_xO)Fe^III OFe^IV] fragment (T model), and does not contain α-hydroxo bridge. When combined with a subsequent ¹⁷O ENDOR study of X prepared with H₂¹⁷O and with ¹⁷O₂, the results led us to suggest that this fragment Is the entire Inorganic core of X. This has been questioned by recent reports, but these reports do not themselves agree on the core of X. One concluded that X possesses a di-μ-oxo Fe^III/Fe^IV core plus a terminal (H₂O) bound to Fe^III [e.g., Han, W.-G.; Liu, T.; Lovell, T.; Noodleman, L. J. Am. Chem. Soc. 2005, 127, 15778-15790]. The other [Mitic, N.; Clay, M. D.; Saleh, L.; Bollinger, J. M.; Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 9049-9065] concluded that X contains only a single oxo bridge and postulated the presence of an additional hydroxo bridge plus a terminal hydroxyl bound to Fe^III. In this report we take advantage of improvements in 35 GHz pulsed ENDOR performance to reexamine the protonation state of oxygenic ligands of the inorganic core of X by directly probing the exchangeable proton(s) with ²H pulsed ENDOR spectroscopy. These ²H ENDOR measurements confirm that X contains an Fe ^III-bound terminal aqua ligand (H_xO), but the spectra contain none of the features that would be required for the proton of a bridging hydroxyl. Thus, we confirm that X contains a terminal aqua (most likely hydroxo) ligand to Fe^III in addition to one or two μ-oxo bridges but does not contain a μ-hydroxo bridge. The ²H ENDOR measurements further demonstrate that this conclusion is applicable to both wild type and Y122F-β2 mutant, and In fact we detect no difference between the properties of protons on the terminal oxygens In the two variants; likewise, ¹⁴N ENDOR measurements of hlstidyl ligands bound to Fe show no difference between the two variants.