The Core Structure of X Generated in the Assembly of the Diiron Cluster of Ribonucleotide Reductase:  17O2 and H2 17O ENDOR

The intermediate, designated X, formed during the self-assembly reaction of the tyrosyl radical/μ-oxo-bridged diferric cofactor in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) is directly involved in the oxidation of Y122 to the catalytically essential ·Y122. Earlier rapid freeze-quench (RFQ) Q-band ENDOR studies led to the formulation of X as a spin-coupled FeIII/FeIV center, with an S = 1/2 ground state, and showed that X contains a single terminal aqua ligand (water molecule or 2-fold disordered hydroxyl) bound to FeIII but does not contain an hydroxyl bridge. That ENDOR data, coupled with RFQ-EXAFS data, plus the strong spin coupling between the iron ions constrain the structure of X to a di- or tribridged species whose inorganic core (defined as iron and exogenous ligands) contains the [(H x O)FeIIIOFeIV] fragment. To determine whether the core contains a second oxo bridge and to establish the fate of the atoms derived from O2, we have now performed CW and pulsed Q-band 17O ENDOR experiments on samples of X prepared in both H2 17O and 17O2, using a uniformly 15N-labeled protein, [U-15N]-R2. These measurements, along with kinetic studies on the formation of X in both wild-type and Y122F R2, as monitored by both ENDOR and S-band EPR spectroscopies, reveal that X contains two oxygen atoms. Both are initially derived from O2, with one present as a μ-oxo bridge and one as the terminal aqua ligand; with time the latter of these atoms exchanges with solvent. These and our previous studies indicate that X does not contain a di-μ-oxo- or μ-oxo,hydroxo-bridged core structure. A structure for X is proposed that contains a single oxo bridge, one terminal aqua ligand bound to the FeIII, and one or two additional mono-oxo bridges provided by the carboxylate oxygens of E115 and/or E238. In addition, the time course of the formation of X in the presence of 17O2 provides important insights into the dynamics of cluster assembly.