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Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Publication ,  Journal Article
Ravichandran, K; Minnihan, EC; Lin, Q; Yokoyama, K; Taguchi, AT; Shao, J; Nocera, DG; Stubbe, J
Published in: Biochemistry
February 14, 2017

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-β2 and 2,3,5-F3Y122•-β2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

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Published In

Biochemistry

DOI

EISSN

1520-4995

Publication Date

February 14, 2017

Volume

56

Issue

6

Start / End Page

856 / 868

Location

United States

Related Subject Headings

  • Tyrosine
  • Ribonucleotide Reductases
  • Recombinant Proteins
  • Protein Interaction Domains and Motifs
  • Protein Conformation
  • Oxidation-Reduction
  • Mutation
  • Mutagenesis, Site-Directed
  • Models, Molecular
  • Kinetics
 

Citation

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Ravichandran, K., Minnihan, E. C., Lin, Q., Yokoyama, K., Taguchi, A. T., Shao, J., … Stubbe, J. (2017). Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase. Biochemistry, 56(6), 856–868. https://doi.org/10.1021/acs.biochem.6b01145
Ravichandran, Kanchana, Ellen C. Minnihan, Qinghui Lin, Kenichi Yokoyama, Alexander T. Taguchi, Jimin Shao, Daniel G. Nocera, and JoAnne Stubbe. “Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.Biochemistry 56, no. 6 (February 14, 2017): 856–68. https://doi.org/10.1021/acs.biochem.6b01145.
Ravichandran K, Minnihan EC, Lin Q, Yokoyama K, Taguchi AT, Shao J, et al. Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase. Biochemistry. 2017 Feb 14;56(6):856–68.
Ravichandran, Kanchana, et al. “Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.Biochemistry, vol. 56, no. 6, Feb. 2017, pp. 856–68. Pubmed, doi:10.1021/acs.biochem.6b01145.
Ravichandran K, Minnihan EC, Lin Q, Yokoyama K, Taguchi AT, Shao J, Nocera DG, Stubbe J. Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase. Biochemistry. 2017 Feb 14;56(6):856–868.
Journal cover image

Published In

Biochemistry

DOI

EISSN

1520-4995

Publication Date

February 14, 2017

Volume

56

Issue

6

Start / End Page

856 / 868

Location

United States

Related Subject Headings

  • Tyrosine
  • Ribonucleotide Reductases
  • Recombinant Proteins
  • Protein Interaction Domains and Motifs
  • Protein Conformation
  • Oxidation-Reduction
  • Mutation
  • Mutagenesis, Site-Directed
  • Models, Molecular
  • Kinetics