Importance of the maintenance pathway in the regulation of the activity of Escherichia coli ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class la RNR is composed of alpha and beta subunits that form an alpha(2)beta(2) active complex. beta contains the diferric tyrosyl radical (Y-center dot) cofactor that is essential for the reduction process that occurs on alpha. [Y-center dot] 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 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, 01 could be varied from 4 to 3300 mu M. [Y-center dot] in vivo and on affinity-purified Strep-P in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1-0.3 Y-center dot radical per P. To determine if the substoichiometric Y-center dot level was associated with apo or diferric beta, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titratiom, followed by addition Of 02 to assemble the cofactor and EPR analysis to quantitate Y-center dot revealed that P is completely loaded with a diferric cluster even when its concentration in vivo is 244 mu M. These titrations, furthermore, resulted in 1 Y-center dot radical per, the highest levels reported. Whole cell Mossbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in beta. These results suggest that modulation of the level of Y-center dot in vivo in E. coli is a mechanism of regulating RNR activity.