The core structure of X generated in the assembly of the diiron cluster ofribonucleotide reductase: O-17(2) and (H2O)-O-17 ENDOR

The intermediate, designated X, formed during the self-assembly reaction of the tyrosyl radical/mu-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 free ze-quench (RFQ) Q-band ENDOR studies led to the formulation of X as a spin- coupled Fe-III/Fe-IV center, with an S = 1/2 ground state, and showed that X contains a single terminal aqua ligand (water molecule or 2-fold disorder ed hydroxyl) bound to Fe-III but does not contain an hydroxyl bridge. That ENDOR data, coupled with RFQ-EXAFS data, plus the strong spin coupling betw een the iron ions constrain the structure of X to a di- or tribridged speci es whose inorganic core (defined as iron and exogenous ligands) contains th e [(HxO)(FeOFeIV)-O-III] fragment. To determine whether the core contains a second oxo bridge and to establish the fate of the atoms derived from O-2, we have now performed CW and pulsed Q-band O-17 ENDOR experiments on sampl es of X prepared in both (H2O)-O-17 and O-17(2), using a uniformly N-15-lab eled protein, [U-N-15]-R2. These measurements, along with kinetic studies o n the formation of X in both wild-type and Y122F R2, as monitored by both E NDOR and S-band EPR spectroscopies, reveal that ii contains two oxygen atom s. Both are initially derived from O-2, with one present as a mu-oxo bridge and one as the terminal aqua ligand; with time the latter of these atoms e xchanges with solvent. These and our previous studies indicate that X does not contain a di-mu-oxo- or mu-oxo,hydroxo-bridged core structure. A struct ure for X is proposed that contains a single oxo bridge, one terminal aqua ligand bound to the Fem, and one or two additional mono-ore bridges provide d by the carboxylate oxygens of E115 and/or E238. In addition, the time cou rse of the formation of X in the presence of O-17(2) provides important ins ights into the dynamics of cluster assembly.