Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants; implications for the O2 activation mechanism

Shen Lu, Eduardo Libby, Lana Saleh, Gang Xing, J. Martin Bollinger, Pierre Moenne-Loccoz

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O2 to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [FeIII-FeIV] state called compound X, but in related diiron proteins such as methane monooxygenase, Δ9-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate μ-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O2 activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/ D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}7]2 center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}7 dimer where both Fe-N-O units share a common vibrational signature. In the wt protein, v(Fe-NO), δ(Fe-N-O), and v(N-O) bands are observed at 445, 434 and 1742 cm-1, respectively, while in the variant proteins the v(Fe-NO) and δ(Fe-N-O) bands are observed ∼10 cm-1 higher and the v(N-O) ∼10 cm-1 lower at 1735 cm-1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}7]2 species with two identical Fe-N-O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O2 activation process in R2-wt.

Original languageEnglish (US)
Pages (from-to)818-827
Number of pages10
JournalJournal of Biological Inorganic Chemistry
Volume9
Issue number7
DOIs
StatePublished - Oct 2004

Fingerprint

Ribonucleotide Reductases
Escherichia coli Proteins
Escherichia coli
Chemical activation
Proteins
methane monooxygenase
Iron
Reaction intermediates
Vibrational spectroscopy
Ferritins
Dimers
Catalytic Domain
Spectrum Analysis
Substitution reactions
Ligands

Keywords

  • Iron-nitrosyl complexes
  • Peroxo-diiron complexes
  • Ribonucleotide reductase
  • Vibrational bands

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants; implications for the O2 activation mechanism. / Lu, Shen; Libby, Eduardo; Saleh, Lana; Xing, Gang; Bollinger, J. Martin; Moenne-Loccoz, Pierre.

In: Journal of Biological Inorganic Chemistry, Vol. 9, No. 7, 10.2004, p. 818-827.

Research output: Contribution to journalArticle

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abstract = "The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O2 to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [FeIII-FeIV] state called compound X, but in related diiron proteins such as methane monooxygenase, Δ9-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate μ-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O2 activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/ D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}7]2 center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}7 dimer where both Fe-N-O units share a common vibrational signature. In the wt protein, v(Fe-NO), δ(Fe-N-O), and v(N-O) bands are observed at 445, 434 and 1742 cm-1, respectively, while in the variant proteins the v(Fe-NO) and δ(Fe-N-O) bands are observed ∼10 cm-1 higher and the v(N-O) ∼10 cm-1 lower at 1735 cm-1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}7]2 species with two identical Fe-N-O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O2 activation process in R2-wt.",
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AU - Xing, Gang

AU - Bollinger, J. Martin

AU - Moenne-Loccoz, Pierre

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N2 - The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O2 to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [FeIII-FeIV] state called compound X, but in related diiron proteins such as methane monooxygenase, Δ9-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate μ-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O2 activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/ D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}7]2 center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}7 dimer where both Fe-N-O units share a common vibrational signature. In the wt protein, v(Fe-NO), δ(Fe-N-O), and v(N-O) bands are observed at 445, 434 and 1742 cm-1, respectively, while in the variant proteins the v(Fe-NO) and δ(Fe-N-O) bands are observed ∼10 cm-1 higher and the v(N-O) ∼10 cm-1 lower at 1735 cm-1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}7]2 species with two identical Fe-N-O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O2 activation process in R2-wt.

AB - The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O2 to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [FeIII-FeIV] state called compound X, but in related diiron proteins such as methane monooxygenase, Δ9-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate μ-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O2 activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/ D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}7]2 center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}7 dimer where both Fe-N-O units share a common vibrational signature. In the wt protein, v(Fe-NO), δ(Fe-N-O), and v(N-O) bands are observed at 445, 434 and 1742 cm-1, respectively, while in the variant proteins the v(Fe-NO) and δ(Fe-N-O) bands are observed ∼10 cm-1 higher and the v(N-O) ∼10 cm-1 lower at 1735 cm-1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}7]2 species with two identical Fe-N-O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O2 activation process in R2-wt.

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