Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of a 1:1 complex of two homodimeric subunits: α and β. β contains the diferric-tyrosyl radical (Y•) cofactor essential for the reduction process. In vivo, the mechanism of Y• regeneration from the diferric-β2 (met-β2) or apo-β2 is still unclear. Y• regenerations from met-β2 and apo-β2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding α and β) were examined. In 29% of the cases, an open reading frame annotated 2Fe2S ferredoxin (YfaE in Escherichia coli) is located next to nrdB. Thus, YfaE has been cloned, expressed, resolubilized, reconstituted anaerobically with Fe 2+, Fe3+, and S2-, and characterized by Mössbauer, EPR, and visible spectroscopies. Titration of met-β2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-β2 and [2Fe2S]2+-YfaE with one Fe reduced/YfaE oxidized. At the end point of the titration, O2 is added to the mixture and the diferrous-β2 rapidly undergoes reaction to form the diferric-Y• with a stoichiometry of 2Fe/Y• and a specific activity correlated to the amount of Y•. The reducing equivalent required for diferric-Y• cofactor biosynthesis is supplied by β. Under anaerobic conditions, stopped flow kinetics have been used to monitor the disappearance of the diferric cluster and the formation of [2Fe2S]2+-YfaE. The titrations and kinetic studies provide the first evidence for a protein involved in the maintenance pathway and likely the biosynthetic pathway.
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