Mechanism of Assembly of the Tyrosyl Radical-Dinuclear Iron Cluster Cofactor of Ribonucleotide Reductase

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Abstract
Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe 2+ and O 2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a μ-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57 Fe 2+ , this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe 2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (⋅Y122) suggest that both intermediates can generate ⋅Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.