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Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 29, 26852-26859 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2001. Vol. 276, no 29, 26852-26859 p.
Keyword [en]
Azides/*metabolism, Electron Spin Resonance Spectroscopy, Escherichia coli/*enzymology, Free Radicals, Iron/*metabolism, Mutagenesis, Protein Binding, Ribonucleotide Reductases/chemistry/genetics/*metabolism, Substrate Specificity, Tyrosine/metabolism
URN: urn:nbn:se:su:diva-19867DOI: 10.1074/jbc.M008190200PubMedID: 11328804OAI: diva2:186391
Available from: 2007-11-20 Created: 2007-11-20 Last updated: 2010-05-07Bibliographically approved
In thesis
1. Biophysical investigations of ribonucleotide reductase: Activation and inhibition mechanisms
Open this publication in new window or tab >>Biophysical investigations of ribonucleotide reductase: Activation and inhibition mechanisms
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribonucleotide reductase (RNR) is the enzyme responsible for de novo synthesis of deoxyribonucleotides, needed for both synthesis and repair of cellular DNA. The RNRs known so far are divided into three distinct classes; I, II and III. The conventional class I enzyme is composed of two separate subunits. The larger R1 subunit contains the active site, whereas the smaller R2 subunit contains a system specialized in forming, transporting and stabilizing a tyrosyl free radical.

Recently a new class Ic RNR was discovered in the bacterium Chlamydia trachomatis. It differs from the conventional class Ia and b RNRs in that it has a phenylalanine at the otherwise conserved tyrosyl radical harboring residue in its R2 subunit. Additionally the metal cluster shows some unusual aspects, of which the most striking perhaps is that the most red-ox active form is a mixed Mn-Fe cluster, instead of the normal Fe-Fe counterpart.

In this work several biochemical and biophysical methods were used to study activation and inhibition mechanisms in RNR from various class I species. The results from studying the oxygen activation confirm the role of the iron ligand E238 as a key residue for controlling the outcome of the reaction in E. coli protein R2. The finding of a stable sulfinyl radical after reconstitution of the R2 Y177F/I263C variant from mouse indicates that sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important in the radical chemistry of RNR. The investigation of the role of the proposed radical transfer pathway during chemical reduction of the iron/radical center shows that no specific pathway is required for the reduction of protein R2 from mouse. The results from inhibition studies of C. trachomatis demonstrate that the same mechanism of inhibition functions on this new class Ic RNR, however less efficiently than in class Ia and b.


Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 52 p.
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urn:nbn:se:su:diva-39043 (URN)978-91-7447-088-8 (ISBN)
Public defence
2010-06-10, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.Available from: 2010-05-19 Created: 2010-05-06 Last updated: 2010-05-07Bibliographically approved

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