Structural Characterization of the Peroxodiiron(III) Intermediate Generated during Oxygen Activation by the W48A/D84E Variant of Ribonucleotide Reductase Protein R2 from Escherichia coli

Jeffrey Baldwin, Carsten Krebs, Lana Saleh, Mindy Stelling, Boi Hanh Huynh, Joseph M. Bollinger, Jr., Pamela Riggs-Gelasco

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a μ-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a μ-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mössbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 Å was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a μ-(η 12) or a μ-1.1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 Å) than does a sample of anaerobically prepared diferrous R2 (3.4 Å). Possible explanations for this difference in detected Fe-Fe distance include an O2-induced conformational change prior to covalent chemistry or differing O2 reactivity among multiple diiron(II) forms of the cluster.

Original languageEnglish (US)
Pages (from-to)13269-13279
Number of pages11
JournalBiochemistry
Volume42
Issue number45
DOIs
StatePublished - Nov 18 2003

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methane monooxygenase
Ribonucleotide Reductases
Escherichia coli Proteins
Escherichia coli
Chemical activation
Oxygen
Catalytic Domain
X-Ray Absorption Spectroscopy
Deoxyribonucleotides
Ribonucleotides
Proteins
X ray absorption spectroscopy
Aspartic Acid
Conformations
Glutamic Acid
Substitution reactions
Ligands
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{22aa1327fb0c4b66bebe0f7b199ac0cf,
title = "Structural Characterization of the Peroxodiiron(III) Intermediate Generated during Oxygen Activation by the W48A/D84E Variant of Ribonucleotide Reductase Protein R2 from Escherichia coli",
abstract = "The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a μ-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a μ-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M{\"o}ssbauer and X-ray absorption spectroscopy. These samples contained 70{\%} peroxodiiron(III) intermediate and 30{\%} diferrous R2. An Fe-Fe distance of 2.5 {\AA} was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a μ-(η 1,η2) or a μ-1.1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 {\AA}) than does a sample of anaerobically prepared diferrous R2 (3.4 {\AA}). Possible explanations for this difference in detected Fe-Fe distance include an O2-induced conformational change prior to covalent chemistry or differing O2 reactivity among multiple diiron(II) forms of the cluster.",
author = "Jeffrey Baldwin and Carsten Krebs and Lana Saleh and Mindy Stelling and Huynh, {Boi Hanh} and {Bollinger, Jr.}, {Joseph M.} and Pamela Riggs-Gelasco",
year = "2003",
month = "11",
day = "18",
doi = "10.1021/bi035198p",
language = "English (US)",
volume = "42",
pages = "13269--13279",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "45",

}

Structural Characterization of the Peroxodiiron(III) Intermediate Generated during Oxygen Activation by the W48A/D84E Variant of Ribonucleotide Reductase Protein R2 from Escherichia coli. / Baldwin, Jeffrey; Krebs, Carsten; Saleh, Lana; Stelling, Mindy; Huynh, Boi Hanh; Bollinger, Jr., Joseph M.; Riggs-Gelasco, Pamela.

In: Biochemistry, Vol. 42, No. 45, 18.11.2003, p. 13269-13279.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structural Characterization of the Peroxodiiron(III) Intermediate Generated during Oxygen Activation by the W48A/D84E Variant of Ribonucleotide Reductase Protein R2 from Escherichia coli

AU - Baldwin, Jeffrey

AU - Krebs, Carsten

AU - Saleh, Lana

AU - Stelling, Mindy

AU - Huynh, Boi Hanh

AU - Bollinger, Jr., Joseph M.

AU - Riggs-Gelasco, Pamela

PY - 2003/11/18

Y1 - 2003/11/18

N2 - The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a μ-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a μ-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mössbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 Å was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a μ-(η 1,η2) or a μ-1.1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 Å) than does a sample of anaerobically prepared diferrous R2 (3.4 Å). Possible explanations for this difference in detected Fe-Fe distance include an O2-induced conformational change prior to covalent chemistry or differing O2 reactivity among multiple diiron(II) forms of the cluster.

AB - The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a μ-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a μ-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mössbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 Å was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a μ-(η 1,η2) or a μ-1.1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 Å) than does a sample of anaerobically prepared diferrous R2 (3.4 Å). Possible explanations for this difference in detected Fe-Fe distance include an O2-induced conformational change prior to covalent chemistry or differing O2 reactivity among multiple diiron(II) forms of the cluster.

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