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: alpha and beta. beta contains the diferric-tyrosyl radical (Y-center dot) cofactor essential for the reduction process. In vivo, the mechanism of Y-center dot regeneration from the diferric-beta(2) (met-beta(2)) or apo-beta(2) is still unclear. Y-center dot regenerations from met-beta(2) and apo-beta(2) have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding alpha and beta) 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 Fe2+, Fe3+, and S2-, and characterized by Mossbauer, EPR, and visible spectroscopies. Titration of met-beta(2) with [2Fe2S](1+)-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-beta(2) and [2Fe2S](2+)-YfaE with one Fe reduced/YfaE oxidized. At the end point of the titration, O-2 is added to the mixture and the diferrous-beta(2) rapidly undergoes reaction to form the diferric-Y-center dot with a stoichiometry of 2Fe/Y-center dot and a specific activity correlated to the amount of Y-center dot. The reducing equivalent required for diferric-Y-center dot cofactor biosynthesis is supplied by beta. 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.