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A new tyrosyl radical on Phe(208) as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H - Combined X-ray diffraction and EPR/ENDOR studies

Kolberg, M ; Logan, Derek LU orcid ; Bleifuss, G ; Potsch, S ; Sjoberg, B-M ; Gräslund, A ; Lubitz, W ; Lassmann, G and Lendzian, F (2005) In Journal of Biological Chemistry 280(12). p.11233-11246
Abstract
The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled... (More)
The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, (FeFeR)-Fe-III-R-III . Here we report a detailed characterization of center H, using H-1/H-2-N-14/N-15- and Fe-57-ENDOR in comparison with the (FeFeIV)-Fe-III intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe(208) is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe(208) is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical. (Less)
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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
280
issue
12
pages
11233 - 11246
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000227761800044
  • scopus:15744363736
  • pmid:15634667
ISSN
1083-351X
DOI
10.1074/jbc.M414634200
language
English
LU publication?
yes
id
0d4933ec-52de-4b07-a803-2cf827cb153b (old id 152270)
date added to LUP
2016-04-01 12:21:42
date last changed
2022-01-27 02:43:05
@article{0d4933ec-52de-4b07-a803-2cf827cb153b,
  abstract     = {{The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, (FeFeR)-Fe-III-R-III . Here we report a detailed characterization of center H, using H-1/H-2-N-14/N-15- and Fe-57-ENDOR in comparison with the (FeFeIV)-Fe-III intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe(208) is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe(208) is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.}},
  author       = {{Kolberg, M and Logan, Derek and Bleifuss, G and Potsch, S and Sjoberg, B-M and Gräslund, A and Lubitz, W and Lassmann, G and Lendzian, F}},
  issn         = {{1083-351X}},
  language     = {{eng}},
  number       = {{12}},
  pages        = {{11233--11246}},
  publisher    = {{American Society for Biochemistry and Molecular Biology}},
  series       = {{Journal of Biological Chemistry}},
  title        = {{A new tyrosyl radical on Phe(208) as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H - Combined X-ray diffraction and EPR/ENDOR studies}},
  url          = {{http://dx.doi.org/10.1074/jbc.M414634200}},
  doi          = {{10.1074/jbc.M414634200}},
  volume       = {{280}},
  year         = {{2005}},
}