Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer

Bigna Wörsdörfer, Denise A. Conner, Kenichi Yokoyama, Jovan Livada, Mohammad Seyedsayamdost, Wei Jiang, Alexey Silakov, Joanne Stubbe, J. Martin Bollinger, Carsten Krebs

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122 by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y 122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y 122, but also directly in catalysis.

Original languageEnglish (US)
Pages (from-to)8585-8593
Number of pages9
JournalJournal of the American Chemical Society
Volume135
Issue number23
DOIs
StatePublished - Jun 12 2013

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Ribonucleotide Reductases
Proton transfer
Escherichia coli
Protons
Amino acids
Ligands
Electrons
Amino Acids
Deprotonation
Water
Free radicals
Catalysis
Free Radicals
Density functional theory
Cysteine
Enzymes
Catalytic Domain
Oxidoreductases

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Wörsdörfer, Bigna ; Conner, Denise A. ; Yokoyama, Kenichi ; Livada, Jovan ; Seyedsayamdost, Mohammad ; Jiang, Wei ; Silakov, Alexey ; Stubbe, Joanne ; Bollinger, J. Martin ; Krebs, Carsten. / Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 23. pp. 8585-8593.
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title = "Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer",
abstract = "The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or {"}hole{"} over a distance of ∼35 {\AA} from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep {"}hopping{"} mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y 122. Here we show that forward radical translocation is associated with perturbation of the M{\"o}ssbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y 122•, but also directly in catalysis.",
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Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer. / Wörsdörfer, Bigna; Conner, Denise A.; Yokoyama, Kenichi; Livada, Jovan; Seyedsayamdost, Mohammad; Jiang, Wei; Silakov, Alexey; Stubbe, Joanne; Bollinger, J. Martin; Krebs, Carsten.

In: Journal of the American Chemical Society, Vol. 135, No. 23, 12.06.2013, p. 8585-8593.

Research output: Contribution to journalArticle

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T1 - Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer

AU - Wörsdörfer, Bigna

AU - Conner, Denise A.

AU - Yokoyama, Kenichi

AU - Livada, Jovan

AU - Seyedsayamdost, Mohammad

AU - Jiang, Wei

AU - Silakov, Alexey

AU - Stubbe, Joanne

AU - Bollinger, J. Martin

AU - Krebs, Carsten

PY - 2013/6/12

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N2 - The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y 122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y 122•, but also directly in catalysis.

AB - The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y 122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y 122•, but also directly in catalysis.

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