A 2.8 Å Fe-Fe separation in the Fe 2 III/IV intermediate, X, from Escherichia coli ribonucleotide reductase

Laura M.K. Dassama, Alexey Silakov, Courtney M. Krest, Julio C. Calixto, Carsten Krebs, Joseph M. Bollinger, Jr., Michael T. Green

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe 2 III/III /tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe 2 II/II complex with O 2 results in accumulation of an Fe 2 III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y 122 ) to the radical (Y 122 ) as the cluster is converted to the μ-oxo-Fe 2 III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d Fe-Fe ) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O 2- , HO - , and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d Fe-Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O 2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d Fe-Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo) 2 -Fe 2 III/IV core. Correction of the d Fe-Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y 122 .

Original languageEnglish (US)
Pages (from-to)16758-16761
Number of pages4
JournalJournal of the American Chemical Society
Volume135
Issue number45
DOIs
StatePublished - Nov 13 2013

Fingerprint

Ribonucleotide Reductases
X ray absorption
Escherichia coli
chlorite dismutase
X-Rays
Cysteine
Enzymes
Extended X ray absorption fine structure spectroscopy
Self assembly
Tyrosine
Iron
Proteins
Spectrum Analysis
Atoms
Substrates
Oxidoreductases

All Science Journal Classification (ASJC) codes

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

Cite this

@article{cd5700dbdfcd48578b78c25914b640ee,
title = "A 2.8 {\AA} Fe-Fe separation in the Fe 2 III/IV intermediate, X, from Escherichia coli ribonucleotide reductase",
abstract = "A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe 2 III/III /tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 {\AA} away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe 2 II/II complex with O 2 results in accumulation of an Fe 2 III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y 122 ) to the radical (Y 122 • ) as the cluster is converted to the μ-oxo-Fe 2 III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d Fe-Fe ) of 2.5 {\AA}, which was interpreted to imply the presence of three single-atom bridges (O 2- , HO - , and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d Fe-Fe ≥ 2.7 {\AA}. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O 2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d Fe-Fe = 2.78 {\AA} is fully consistent with computational models containing a (μ-oxo) 2 -Fe 2 III/IV core. Correction of the d Fe-Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y 122 • .",
author = "Dassama, {Laura M.K.} and Alexey Silakov and Krest, {Courtney M.} and Calixto, {Julio C.} and Carsten Krebs and {Bollinger, Jr.}, {Joseph M.} and Green, {Michael T.}",
year = "2013",
month = "11",
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pages = "16758--16761",
journal = "Journal of the American Chemical Society",
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A 2.8 Å Fe-Fe separation in the Fe 2 III/IV intermediate, X, from Escherichia coli ribonucleotide reductase . / Dassama, Laura M.K.; Silakov, Alexey; Krest, Courtney M.; Calixto, Julio C.; Krebs, Carsten; Bollinger, Jr., Joseph M.; Green, Michael T.

In: Journal of the American Chemical Society, Vol. 135, No. 45, 13.11.2013, p. 16758-16761.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A 2.8 Å Fe-Fe separation in the Fe 2 III/IV intermediate, X, from Escherichia coli ribonucleotide reductase

AU - Dassama, Laura M.K.

AU - Silakov, Alexey

AU - Krest, Courtney M.

AU - Calixto, Julio C.

AU - Krebs, Carsten

AU - Bollinger, Jr., Joseph M.

AU - Green, Michael T.

PY - 2013/11/13

Y1 - 2013/11/13

N2 - A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe 2 III/III /tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe 2 II/II complex with O 2 results in accumulation of an Fe 2 III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y 122 ) to the radical (Y 122 • ) as the cluster is converted to the μ-oxo-Fe 2 III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d Fe-Fe ) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O 2- , HO - , and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d Fe-Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O 2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d Fe-Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo) 2 -Fe 2 III/IV core. Correction of the d Fe-Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y 122 • .

AB - A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe 2 III/III /tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe 2 II/II complex with O 2 results in accumulation of an Fe 2 III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y 122 ) to the radical (Y 122 • ) as the cluster is converted to the μ-oxo-Fe 2 III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d Fe-Fe ) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O 2- , HO - , and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d Fe-Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O 2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d Fe-Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo) 2 -Fe 2 III/IV core. Correction of the d Fe-Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y 122 • .

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