Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction i...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-07, Vol.276 (29), p.26852-26859
Main Authors: Assarsson, M, Andersson, M E, Högbom, M, Persson, B O, Sahlin, M, Barra, A L, Sjöberg, B M, Nordlund, P, Gräslund, A
Format: Article
Language:English
Subjects:
AR2 protein
azide
Azides - metabolism
Electron Spin Resonance Spectroscopy
Escherichia coli
Escherichia coli - enzymology
Free Radicals
Iron - metabolism
Mutagenesis
Protein Binding
Ribonucleotide Reductases - chemistry
Ribonucleotide Reductases - genetics
Ribonucleotide Reductases - metabolism
Ribonucleotide Reductases/chemistry/genetics/metabolism
Substrate Specificity
Tyrosine - metabolism
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Summary:The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M008190200