Two-Color Valence-to-Core X-ray Emission Spectroscopy Tracks Cofactor Protonation State in a Class I Ribonucleotide Reductase

Proton transfer reactions are of central importance to a wide variety of biochemical processes, though determining proton location and monitoring proton transfers in biological systems is often extremely challenging. Herein, we use two-color valence-to-core X-ray emission spectroscopy (VtC XES) to identify protonation events across three oxidation states of the O-2-activating, radical-initiating manganese-iron heterodinuclear cofactor in a class I-c ribonucleotide reductase. This is the first application of VtC XES to an enzyme intermediate and the first simultaneous measurement of two-color VtC spectra. In contrast to more conventional methods of assessing protonation state, VtC XES is a more direct probe applicable to a wide range of metalloenzyme systems. These data, coupled to insight provided by DFT calculations, allow the inorganic cores of the (MnFeIV)-Fe-IV and (MnFeIII)-Fe-IV states of the enzyme to be assigned as Mn-IV(-O)(2)Fe-IV and Mn-IV(-O)(-OH)Fe-III, respectively.