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Title: Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase

Abstract

The outcome of O{sub 2} activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine){sub 2}-(carboxylate){sub 4} ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a {mu}-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H{sub peroxo}) in O{sub 2} activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the 'extra' electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the {mu}-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 and which converts very slowly (t{sub 1/2} {approx} 7 h) upon incubation at 0 C to an intensely purple final product. X-ray crystallographic analysis of the purple productmore » indicates that F208 has undergone {epsilon}-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with {sup 16}O{sub 2} or {sub 18}O{sub 2} show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O{sub 2}. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Moessbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which {mu}-O{sup 2-} is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little {epsilon}-hydroxyphenylalanine is formed and pathways leading to Y122 formation predominate in both R2-D84E and R2-W48F.« less

Authors:
; ; ; ; ; ; ; ; ;  [1]
  1. (NWU)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1006347
Resource Type:
Journal Article
Journal Name:
J. Am. Chem. Soc.
Additional Journal Information:
Journal Volume: 123; Journal Issue: 2001; Journal ID: ISSN 0002-7863
Country of Publication:
United States
Language:
ENGLISH
Subject:
03 NATURAL GAS; AMINO ACIDS; CHEMICAL ANALYSIS; DECAY; ELECTRONS; ESCHERICHIA COLI; HYDROXYLASES; HYDROXYLATION; INCUBATION; IRON; METHANE; OXIDOREDUCTASES; OXYGEN; PHENOL; PHENOLATES; PHENYLALANINE; PROTEINS; RADICALS; RESIDUES; RESONANCE; SPECTRA

Citation Formats

Baldwin, J., Voegtli, W.C., Khidekel, N., Moënne-Loccoz, P., Krebs, C., Ley, B.A., Huynh, B.H., Loehr, T.M., Rosenzweig, A.C., and Bollinger, Jr., J.M. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase. United States: N. p., 2010. Web.
Baldwin, J., Voegtli, W.C., Khidekel, N., Moënne-Loccoz, P., Krebs, C., Ley, B.A., Huynh, B.H., Loehr, T.M., Rosenzweig, A.C., & Bollinger, Jr., J.M. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase. United States.
Baldwin, J., Voegtli, W.C., Khidekel, N., Moënne-Loccoz, P., Krebs, C., Ley, B.A., Huynh, B.H., Loehr, T.M., Rosenzweig, A.C., and Bollinger, Jr., J.M. Fri . "Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase". United States.
@article{osti_1006347,
title = {Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase},
author = {Baldwin, J. and Voegtli, W.C. and Khidekel, N. and Moënne-Loccoz, P. and Krebs, C. and Ley, B.A. and Huynh, B.H. and Loehr, T.M. and Rosenzweig, A.C. and Bollinger, Jr., J.M.},
abstractNote = {The outcome of O{sub 2} activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine){sub 2}-(carboxylate){sub 4} ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a {mu}-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H{sub peroxo}) in O{sub 2} activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the 'extra' electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the {mu}-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 and which converts very slowly (t{sub 1/2} {approx} 7 h) upon incubation at 0 C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone {epsilon}-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with {sup 16}O{sub 2} or {sub 18}O{sub 2} show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O{sub 2}. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Moessbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which {mu}-O{sup 2-} is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little {epsilon}-hydroxyphenylalanine is formed and pathways leading to Y122 formation predominate in both R2-D84E and R2-W48F.},
doi = {},
journal = {J. Am. Chem. Soc.},
issn = {0002-7863},
number = 2001,
volume = 123,
place = {United States},
year = {2010},
month = {3}
}