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Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading

Rozman Grinberg, Inna; Berglund, Sigrid; Hasan, Mahmudul LU ; Lundin, Daniel; Ho, Felix M.; Magnuson, Ann LU ; Logan, Derek T. LU ; Sjöberg, Britt Marie and Berggren, Gustav (2019) In Journal of Biological Inorganic Chemistry
Abstract

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and... (More)

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2 ·−). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.

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author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
Dimanganese cofactor, Electron paramagnetic resonance, Phylogeny, Radicals, Ribonucleotide reductase, X-ray crystallography
in
Journal of Biological Inorganic Chemistry
publisher
Springer
external identifiers
  • scopus:85070994147
ISSN
0949-8257
DOI
10.1007/s00775-019-01697-8
language
English
LU publication?
yes
id
1b74888f-254c-4a65-a712-d3a5ff6e1f60
date added to LUP
2019-09-09 10:32:03
date last changed
2019-09-26 04:41:47
@article{1b74888f-254c-4a65-a712-d3a5ff6e1f60,
  abstract     = {<p>Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn<sub>2</sub>(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn<sub>2</sub>(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn<sub>2</sub>(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H<sub>2</sub>O<sub>2</sub> and superoxide (O<sub>2</sub> <sup>·−</sup>). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.</p>},
  author       = {Rozman Grinberg, Inna and Berglund, Sigrid and Hasan, Mahmudul and Lundin, Daniel and Ho, Felix M. and Magnuson, Ann and Logan, Derek T. and Sjöberg, Britt Marie and Berggren, Gustav},
  issn         = {0949-8257},
  keyword      = {Dimanganese cofactor,Electron paramagnetic resonance,Phylogeny,Radicals,Ribonucleotide reductase,X-ray crystallography},
  language     = {eng},
  publisher    = {Springer},
  series       = {Journal of Biological Inorganic Chemistry},
  title        = {Class Id ribonucleotide reductase utilizes a Mn<sub>2</sub>(IV,III) cofactor and undergoes large conformational changes on metal loading},
  url          = {http://dx.doi.org/10.1007/s00775-019-01697-8},
  year         = {2019},
}