YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase

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 S^2-, 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.