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 A. Cotruvo; Troy A. Stich; R. David Britt; Joanne Stubbe

doi:10.1021/ja312457t

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 A. Cotruvo
, Troy A. Stich
, R. David Britt
, Joanne Stubbe

Research output: Contribution to journal › Article › peer-review

90 Scopus citations

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 Fe^III ₂-Y^• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe^II₂-NrdB, O₂, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn^III₂-Y ^• cofactor in their NrdF subunit. Mn^II ₂-NrdF does not react with O₂, but it binds the reduced form of a conserved flavodoxin-like protein, NrdI_hq, which, in the presence of O₂, reacts to form the Mn^III ₂-Y^• cofactor. Here we investigate the mechanism of assembly of the Mn^III₂-Y^• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn^II₂-NrdF, NrdI_hq, and O₂ has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI_hq reduces O₂ to O₂^•- (40-48 s^-1, 0.6 mM O₂), the O₂ ^•- channels to and reacts with Mn^II₂-NrdF to form a Mn^IIIMn^IV intermediate (2.2 ± 0.4 s ^-1), and the Mn^IIIMn^IV species oxidizes tyrosine to Y^• (0.08-0.15 s^-1). Controlled production of O₂^•- by NrdI_hq during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn^II ₂ cluster with O₂ and satisfies the requirement for an "extra" reducing equivalent in Y^• generation.

Original language	English (US)
Pages (from-to)	4027-4039
Number of pages	13
Journal	Journal of the American Chemical Society
Volume	135
Issue number	10
DOIs	https://doi.org/10.1021/ja312457t
State	Published - Mar 13 2013

All Science Journal Classification (ASJC) codes

Catalysis
General Chemistry
Biochemistry
Colloid and Surface Chemistry

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@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, \{Joseph A.\} and Stich, \{Troy A.\} and Britt, \{R. David\} and Joanne Stubbe",

year = "2013",

month = mar,

day = "13",

doi = "10.1021/ja312457t",

language = "English (US)",

volume = "135",

pages = "4027--4039",

journal = "Journal of the American Chemical Society",

issn = "0002-7863",

publisher = "American Chemical Society",

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}

Cotruvo, JA, Stich, TA, Britt, RD & Stubbe, J 2013, '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', Journal of the American Chemical Society, vol. 135, no. 10, pp. 4027-4039. https://doi.org/10.1021/ja312457t

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. / Cotruvo, Joseph A.; Stich, Troy A.; Britt, R. David et al.
In: Journal of the American Chemical Society, Vol. 135, No. 10, 13.03.2013, p. 4027-4039.

Research output: Contribution to journal › Article › peer-review

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, Joseph A.

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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UR - https://www.scopus.com/pages/publications/84874987181#tab=citedBy

U2 - 10.1021/ja312457t

DO - 10.1021/ja312457t

M3 - Article

C2 - 23402532

AN - SCOPUS:84874987181

SN - 0002-7863

VL - 135

SP - 4027

EP - 4039

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 10

ER -

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

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