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Chemical reduction of the diferric/radical center in protein R2 from mouse ribonucleotide reductase is independent of the proposed radical transfer pathway
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Astrid Gräslund)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Astrid Gräslund)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Astrid Gräslund)
Department of Medical Biosciences, Medical Biochemistry, Umeå Universtity. (Lars Thelander)
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2002 (English)In: Inorganica Chimica Acta, ISSN 0020-1693, E-ISSN 1873-3255, Vol. 331, no 1, 65-72 p.Article in journal (Refereed) Published
Abstract [en]

The rates of reduction of the diferric/radical center in mouse ribonucleotide reductase protein R2 were studied by light absorption and EPR in the native protein and in three point mutants of conserved residues involved in the proposed radical transfer pathway (D266A, W103Y) or in the unstructured C terminal domain (Y370W). The pseudo-first order rate constants for chemical reduction of the tyrosyl radical and diferric center by hydroxyurea, sodium dithionite or the dihydro form of flavin adenine dinucleotide, were comparable with or higher (particularly D266A, by dithionite) than in native R2. Molecular modeling of the D266A mutant showed that the iron/radical site should be more accessible for external reductants in the mutant than in native R2. The results indicate that no specific pathway is required for the reduction. The dihydro form of flavin adenine dinucleotide was found to be a very efficient reductant in the studied proteins compared to dithionite alone. The EPR spectra of the mixed-valent Fe(II)Fe(III) sites formed by chemical reduction in the D266A and W103Y mutants were clearly different from the spectrum observed in the native protein, indicating that the structure of the diferric site was affected by the mutations, as also suggested by the modeling study. No difference was observed between the mixed-valent EPR spectra generated by chemical reduction in Y370W mutant and native mouse R2 protein

Place, publisher, year, edition, pages
2002. Vol. 331, no 1, 65-72 p.
Keyword [en]
Ribonucleotide reductase; Tyrosyl radical reduction; Diiron–oxygen protein
Identifiers
URN: urn:nbn:se:su:diva-39039DOI: 10.1016/S0020-1693(01)00750-2OAI: oai:DiVA.org:su-39039DiVA: diva2:318088
Available from: 2010-05-06 Created: 2010-05-06 Last updated: 2017-12-12Bibliographically 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.
National Category
Biophysics
Research subject
Biophysics
Identifiers
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)
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Note
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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