Identification of the protonated oxygenic ligands of ribonucleotide reductase intermediate X by Q-band ^1,2H CW and pulsed ENDOR

We report CW and pulsed Q-band ^1,2H ENDOR measurements of intermediate X formed during the assembly of the diferric tyrosyl radical cofactor of the R2 subunit in ribonucleotide reductase. These studies, performed with H₂O and D₂O buffers, were designed to determine whether the exchangeable proton signals are associated with an hydroxo bridge, a terminal water, or both. In doing so, we identify the types of protonated oxygen (OH(x)) species coordinated to the iron ions of X and their disposition relative to the ferric and ferryl iron ions. The exchangeable proton signals displayed by intermediate X belong to two protons associated with a terminal water bound to Fe,(III) and not to an hydroxo bridge; within the precision of the modeling, this picture of a terminal water is indistinguishable from that of a 2-fold disordered terminal hydroxyl. The fact that X displays strong spin-coupling between iron ions requires that there be one or more oxo/hydroxo bridges. These findings then establish that X contains the [(H(x)O)Fe(III)OFe(IV)] fragment.