Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (MnIV/FeIV) Chlamydia trachomatis ribonucleotide reductase

Laura M.K. Dassama, Wei Jiang, Paul T. Varano, Maria Eirini Pandelia, Denise A. Conner, Jiajia Xie, Joseph M. Bollinger, Jr., Carsten Krebs

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H) from the substrate. With either oxidant, the inter-subunit "hole- transfer" or "radical-translocation" (RT) process is thought to occur by a "hopping" mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y's. The dependence of Y accumulation on the presence of substrate suggests that RT within this "super-oxidized" enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y's in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the "hurdling" of engineered defects in the RT pathway.

Original languageEnglish (US)
Pages (from-to)20498-20506
Number of pages9
JournalJournal of the American Chemical Society
Volume134
Issue number50
DOIs
StatePublished - Dec 19 2012

Fingerprint

Ribonucleotide Reductases
Chlamydia trachomatis
Defects
Enzymes
Proteins
Hydrogen
Substrates
Nucleotides
Phenylalanine
Oxidants
Cysteine
Paramagnetic resonance
Substitution reactions
Oxidoreductases

All Science Journal Classification (ASJC) codes

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

Cite this

Dassama, Laura M.K. ; Jiang, Wei ; Varano, Paul T. ; Pandelia, Maria Eirini ; Conner, Denise A. ; Xie, Jiajia ; Bollinger, Jr., Joseph M. ; Krebs, Carsten. / Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (MnIV/FeIV) Chlamydia trachomatis ribonucleotide reductase. In: Journal of the American Chemical Society. 2012 ; Vol. 134, No. 50. pp. 20498-20506.
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abstract = "A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y•) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼35 {\AA} away in its α subunit, generating a thiyl radical that abstracts hydrogen (H•) from the substrate. With either oxidant, the inter-subunit {"}hole- transfer{"} or {"}radical-translocation{"} (RT) process is thought to occur by a {"}hopping{"} mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y•'s. The dependence of Y• accumulation on the presence of substrate suggests that RT within this {"}super-oxidized{"} enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y•(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y•'s in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the {"}hurdling{"} of engineered defects in the RT pathway.",
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Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (MnIV/FeIV) Chlamydia trachomatis ribonucleotide reductase. / Dassama, Laura M.K.; Jiang, Wei; Varano, Paul T.; Pandelia, Maria Eirini; Conner, Denise A.; Xie, Jiajia; Bollinger, Jr., Joseph M.; Krebs, Carsten.

In: Journal of the American Chemical Society, Vol. 134, No. 50, 19.12.2012, p. 20498-20506.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (MnIV/FeIV) Chlamydia trachomatis ribonucleotide reductase

AU - Dassama, Laura M.K.

AU - Jiang, Wei

AU - Varano, Paul T.

AU - Pandelia, Maria Eirini

AU - Conner, Denise A.

AU - Xie, Jiajia

AU - Bollinger, Jr., Joseph M.

AU - Krebs, Carsten

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AB - A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y•) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H•) from the substrate. With either oxidant, the inter-subunit "hole- transfer" or "radical-translocation" (RT) process is thought to occur by a "hopping" mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y•'s. The dependence of Y• accumulation on the presence of substrate suggests that RT within this "super-oxidized" enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y•(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y•'s in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the "hurdling" of engineered defects in the RT pathway.

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