YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase

Chia Hung Wu, Wei Jiang, Carsten Krebs, Joanne Stubbe

Research output: Contribution to journalArticle

39 Citations (Scopus)

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: α and β. β contains the diferric-tyrosyl radical (Y) cofactor essential for the reduction process. In vivo, the mechanism of Y regeneration from the diferric-β2 (met-β2) or apo-β2 is still unclear. Y regenerations from met-β2 and apo-β2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding α and β) 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 Fe 2+, Fe3+, and S2-, and characterized by Mössbauer, EPR, and visible spectroscopies. Titration of met-β2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-β2 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-β2 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 β. 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.

Original languageEnglish (US)
Pages (from-to)11577-11588
Number of pages12
JournalBiochemistry
Volume46
Issue number41
DOIs
StatePublished - Oct 16 2007

Fingerprint

Ribonucleotide Reductases
Ferredoxins
Biosynthetic Pathways
Titration
Escherichia coli
Regeneration
Maintenance
Open Reading Frames
Spectrum Analysis
Kinetics
Gene encoding
Nucleotides
Biosynthesis
Genome
Stoichiometry
Paramagnetic resonance
Genes
Spectroscopy
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Wu, Chia Hung ; Jiang, Wei ; Krebs, Carsten ; Stubbe, Joanne. / YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase. In: Biochemistry. 2007 ; Vol. 46, No. 41. pp. 11577-11588.
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title = "YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase",
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: α and β. β contains the diferric-tyrosyl radical (Y•) cofactor essential for the reduction process. In vivo, the mechanism of Y• regeneration from the diferric-β2 (met-β2) or apo-β2 is still unclear. Y• regenerations from met-β2 and apo-β2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding α and β) 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 Fe 2+, Fe3+, and S2-, and characterized by M{\"o}ssbauer, EPR, and visible spectroscopies. Titration of met-β2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-β2 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-β2 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 β. 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.",
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YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase. / Wu, Chia Hung; Jiang, Wei; Krebs, Carsten; Stubbe, Joanne.

In: Biochemistry, Vol. 46, No. 41, 16.10.2007, p. 11577-11588.

Research output: Contribution to journalArticle

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N2 - 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: α and β. β contains the diferric-tyrosyl radical (Y•) cofactor essential for the reduction process. In vivo, the mechanism of Y• regeneration from the diferric-β2 (met-β2) or apo-β2 is still unclear. Y• regenerations from met-β2 and apo-β2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding α and β) 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 Fe 2+, Fe3+, and S2-, and characterized by Mössbauer, EPR, and visible spectroscopies. Titration of met-β2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-β2 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-β2 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 β. 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.

AB - 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: α and β. β contains the diferric-tyrosyl radical (Y•) cofactor essential for the reduction process. In vivo, the mechanism of Y• regeneration from the diferric-β2 (met-β2) or apo-β2 is still unclear. Y• regenerations from met-β2 and apo-β2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding α and β) 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 Fe 2+, Fe3+, and S2-, and characterized by Mössbauer, EPR, and visible spectroscopies. Titration of met-β2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-β2 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-β2 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 β. 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.

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