Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase

Enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate

Joseph Alfred Cotruvo, Jr., Troy A. Stich, R. David Britt, Joanne Stubbe

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y) generated by oxidation of a reduced dinuclear metal cluster. The FeIII 2-Y cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y cofactor in their NrdF subunit. MnII 2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII 2-Y cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•- (40-48 s-1, 0.6 mM O2), the O2 •- channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s -1), and the MnIIIMnIV species oxidizes tyrosine to Y (0.08-0.15 s-1). Controlled production of O2•- by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII 2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y generation.

Original languageEnglish (US)
Pages (from-to)4027-4039
Number of pages13
JournalJournal of the American Chemical Society
Volume135
Issue number10
DOIs
StatePublished - Mar 13 2013

Fingerprint

Ribonucleotide Reductases
Superoxides
Tyrosine
Oxidation
Flavodoxin
Bacilli
Nucleotides
Bacillus subtilis
Self assembly
Paramagnetic resonance
Spectrum Analysis
Enzymes
Metals
Spectroscopy
Proteins
Oxidoreductases

All Science Journal Classification (ASJC) codes

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

Cite this

@article{6b2643b383924cbcbb16f146b62a7fe3,
title = "Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: Enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate",
abstract = "Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The FeIII 2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y • cofactor in their NrdF subunit. MnII 2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII 2-Y• cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•- (40-48 s-1, 0.6 mM O2), the O2 •- channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s -1), and the MnIIIMnIV species oxidizes tyrosine to Y• (0.08-0.15 s-1). Controlled production of O2•- by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII 2 cluster with O2 and satisfies the requirement for an {"}extra{"} reducing equivalent in Y• generation.",
author = "{Cotruvo, Jr.}, {Joseph Alfred} and Stich, {Troy A.} and Britt, {R. David} and Joanne Stubbe",
year = "2013",
month = "3",
day = "13",
doi = "10.1021/ja312457t",
language = "English (US)",
volume = "135",
pages = "4027--4039",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
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TY - JOUR

T1 - Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase

T2 - Enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate

AU - Cotruvo, Jr., Joseph Alfred

AU - Stich, Troy A.

AU - Britt, R. David

AU - Stubbe, Joanne

PY - 2013/3/13

Y1 - 2013/3/13

N2 - Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The FeIII 2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y • cofactor in their NrdF subunit. MnII 2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII 2-Y• cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•- (40-48 s-1, 0.6 mM O2), the O2 •- channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s -1), and the MnIIIMnIV species oxidizes tyrosine to Y• (0.08-0.15 s-1). Controlled production of O2•- by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII 2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y• generation.

AB - Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The FeIII 2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y • cofactor in their NrdF subunit. MnII 2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII 2-Y• cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•- (40-48 s-1, 0.6 mM O2), the O2 •- channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s -1), and the MnIIIMnIV species oxidizes tyrosine to Y• (0.08-0.15 s-1). Controlled production of O2•- by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII 2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y• generation.

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U2 - 10.1021/ja312457t

DO - 10.1021/ja312457t

M3 - Article

VL - 135

SP - 4027

EP - 4039

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 10

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