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EPR studies on a stable sulfinyl radical observed in the iron-oxygen reconstituted Y177F/I263C protein double mutant of ribonucleotide reductase from mouse
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Astrid Gräslund)
(Astrid Gräslund)
The High Field Laboratory, CNRS/MPI, Grenoble, France.
Department of Medical Biochemistry and Biophysicis, Umeå University.
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2002 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 20, 6510-6516 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the μ-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.

Place, publisher, year, edition, pages
2002. Vol. 41, no 20, 6510-6516 p.
URN: urn:nbn:se:su:diva-39041DOI: 10.1021/bi012043dOAI: diva2:318089
Available from: 2010-05-06 Created: 2010-05-06 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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