(μ-1,2-peroxo)diiron(III/IIl) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse

Stopped-flow absorption and freeze-quench electron paramagnetic resonance (EPR) and Mössbauer spectroscopies have been used to obtain evidence for the intermediacy of a (μ-1,2-peroxo)-diiron(III/III) complex on the pathway to the tyrosyl radical and (μ-oxo)diiron(III/III) cluster during assembly of the essential cofactor in the R2 subunit of ribonucleotide reductase from mouse. The complex accumulates to ∼0.4 equiv in the first few milliseconds of the reaction and decays concomitantly with accumulation of the previously detected diiron(III/IV) cluster, X, which generates the tyrosyl radical and product (μ-oxo)diiron(III/III) cluster. Kinetic complexities in the reaction suggest the existence of an anti-cooperative interaction of the monomers of the R2 homodimer in Fe(II) binding and perhaps O₂ activation. The detection of the (μ-1,2-peroxo)diiron(III/III) complex, which has spectroscopic properties similar to those of complexes previously characterized in the reactions of soluble methane monooxygenase, stearoyl acyl carrier protein Δ⁹ desaturase, and variants of Escherichia coli R2 with the iron ligand substitution, D84E, provides support for the hypothesis that the reactions of the diiron-carboxylate oxidases and oxygenases commence with the formation of this common intermediate.