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*) 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.
Wu
Chia-Hung
CH
Departments of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Jiang
Wei
W
Krebs
Carsten
C
Stubbe
JoAnne
J
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer; D014443:Tyrosine / Q000737:chemistry*
D029968:Escherichia coli Proteins; D005288:Ferredoxins; D011994:Recombinant Proteins; C525745:YfaE protein, E coli; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases