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.
The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y • ) cofactor. In Saccharomyces cerevisiae, two R1s, α n and , have been identified, while R2 is a heterodimer (ββ′). β′ cannot bind iron and generate the Y • ; consequently, the maximum amount of Y • per ββ′ is 1. To determine the cofactor stoichiometry in vivo, a FLAGtagged β ( FLAG β) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of FLAG ββ′ by immunoaffinity chromatography, which was found to have 0.45 ± 0.08 Y • / FLAG ββ′ and a specific activity of 2.3 ± 0.5 μmol min −1 mg −1 . FLAG ββ′ isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y • / FLAG ββ′ ratio of 0.5. To determine the Y • /ββ′ ratio without R2 isolation, whole cell EPR and quantitative Western blots of β were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y • /ββ′ ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Δ gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y • /ββ′ ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y • and modulation of Y • concentration is not involved in the regulation of RNR activity.
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