Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase

Lana Saleh, Carsten Krebs, Brenda A. Ley, Sunail Naik, Boi Hanh Huynh, Joseph M. Bollinger, Jr.

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

A key step in generation of the catalytically essential tyrosyl radical (Y122•) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe2O2)4+ complex formed by addition of O 2 to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe2O2)4+ complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48 +•). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe2O2)4+ state that is probably the precursor to the X-W48+• diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O2 results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively μ-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H peroxo) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe2O 2)4+ state in R2-W48A/Y122F. It is postulated that the new (Fe2O2)4+ state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (μ-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.

Original languageEnglish (US)
Pages (from-to)5953-5964
Number of pages12
JournalBiochemistry
Volume43
Issue number20
DOIs
StatePublished - May 25 2004

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Ribonucleotide Reductases
Escherichia coli
Iron
Skatole
Electrons
methane monooxygenase
Isomers
Ligands
Magnetic couplings
Proteins
Escherichia coli Proteins
Peroxides
Tryptophan
Alanine
Electronic structure
Cations
Substitution reactions
Chemical activation
Ions
Oxygen

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase",
abstract = "A key step in generation of the catalytically essential tyrosyl radical (Y122•) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe2O2)4+ complex formed by addition of O 2 to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe2O2)4+ complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a {"}diradical{"} intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48 +•). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe2O2)4+ state that is probably the precursor to the X-W48+• diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O2 results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent M{\"o}ssbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their M{\"o}ssbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or M{\"o}ssbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively μ-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H peroxo) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe2O 2)4+ state in R2-W48A/Y122F. It is postulated that the new (Fe2O2)4+ state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (μ-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.",
author = "Lana Saleh and Carsten Krebs and Ley, {Brenda A.} and Sunail Naik and Huynh, {Boi Hanh} and {Bollinger, Jr.}, {Joseph M.}",
year = "2004",
month = "5",
day = "25",
doi = "10.1021/bi036099e",
language = "English (US)",
volume = "43",
pages = "5953--5964",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
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}

Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase. / Saleh, Lana; Krebs, Carsten; Ley, Brenda A.; Naik, Sunail; Huynh, Boi Hanh; Bollinger, Jr., Joseph M.

In: Biochemistry, Vol. 43, No. 20, 25.05.2004, p. 5953-5964.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase

AU - Saleh, Lana

AU - Krebs, Carsten

AU - Ley, Brenda A.

AU - Naik, Sunail

AU - Huynh, Boi Hanh

AU - Bollinger, Jr., Joseph M.

PY - 2004/5/25

Y1 - 2004/5/25

N2 - A key step in generation of the catalytically essential tyrosyl radical (Y122•) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe2O2)4+ complex formed by addition of O 2 to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe2O2)4+ complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48 +•). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe2O2)4+ state that is probably the precursor to the X-W48+• diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O2 results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively μ-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H peroxo) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe2O 2)4+ state in R2-W48A/Y122F. It is postulated that the new (Fe2O2)4+ state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (μ-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.

AB - A key step in generation of the catalytically essential tyrosyl radical (Y122•) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe2O2)4+ complex formed by addition of O 2 to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe2O2)4+ complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48 +•). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe2O2)4+ state that is probably the precursor to the X-W48+• diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O2 results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively μ-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H peroxo) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe2O 2)4+ state in R2-W48A/Y122F. It is postulated that the new (Fe2O2)4+ state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (μ-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.

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