PMID:17880186
| Citation |
Wu, CH, Jiang, W, Krebs, C and Stubbe, J (2007) YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase. Biochemistry 46:11577-88 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1021/bi7012454 |
| Keywords |
Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Ferredoxins/chemistry; Ferredoxins/genetics; Ferredoxins/metabolism; Genome, Bacterial; Iron/metabolism; Kinetics; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Restriction Mapping; Ribonucleotide Reductases/metabolism; Spectroscopy, Mossbauer; Tyrosine/chemistry |
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