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*) cofactor essential for the reduction process. In vivo, the mechanism of Y* regeneration from the diferric-beta2 (met-beta2) or apo-beta2 is still unclear. Y* regenerations from met-beta2 and apo-beta2 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 Mössbauer, EPR, and visible spectroscopies. Titration of met-beta2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-beta2 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-beta2 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 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.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α2β2 active complex. β contains the diferric tyrosyl radical (Y•) cofactor that is essential for the reduction process that occurs on α. [Y•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an l-arabinose promoter were constructed. Using these constructs and with [l-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 µM. [Y•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1–0.3 Y• radical per β. To determine if the substoichiometric Y• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O2 to assemble the cofactor and EPR analysis to quantitate Y•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 µM. These titrations, furthermore, resulted in 1 Y• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y• in vivo in E. coli is a mechanism of regulating RNR activity.

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