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- Davydov, Albert, 1969-
(author)
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Electron paramagnetic resonance and biochemical studies of red-ox properties of the diferric/radical center in mouse and Mycobacterium tuberculosis ribonucleotide reductase R2 proteins
- 1999
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Doctoral thesis (other academic/artistic)abstract
- Ribonucleotide reductase (RNR) catalyses the reduction of all four ribonucleotides to the corresponding deoxyribonucleotides that are used by the cells for DNA synthesis. The active enzyme from mouse, Mycobacterium tuberculosis (M. tuberculosis) and aerobically grown E. coli consists of two nonidentical subunits, proteins R1 and R2, both required for enzymatic activity. The R1 protein contains the regulatory sites and the binding site for the ribonucleotide substrates whereas the R2 subunit contains a stable tyrosyl radical and an antiferromagnetically coupled _-oxo-bridged diferric center. According to the proposed reaction mechanism the radical properties of the R2 protein are transferred to the active site of the R1 protein upon binding of the substrate molecule to the R1. This transfer is proposed to propagate via a conserved chain of hydrogen-bonded amino acids within the R1 and R2 proteins. Once generated the diferric/radical center may exist in different red-ox states: active (with Fe(III)Fe(III) center and tyrosyl radical), met (Fe(III)Fe(III) center without tyrosyl radical), mixed-valent (Fe(II)Fe(III) center) and fully reduced (Fe(II)Fe(II) center). Admission of oxygen to the fully reduced form of an R2 protein (a so-called regeneration reaction) results in a spontaneous formation of the active form. The regeneration reaction may be one of the possible ways employed by the cell for the generation of the active enzyme and therefore the study of this reaction is important for understanding the enzyme functionality.The aim of this thesis is to study the red-ox transitions of the diferric/radical center in mouse and Mycobacterium tuberculosis R2 proteins. Despite the significant similarities in the structure, the red-ox properties of the diferric/radical centers in mouse and M. tuberculosis R2 proteins are significantly different. The diferric/radical center in mouse R2 was found to be much more accessible for the external reductants than the centers of E. coli and M. tuberculosis R2 proteins. A higher accessibility of the diferric/radical center in mouse R2 protein can be explained by the presence of an open channel from the surface of the protein to the diferric/radical center. The tyrosyl radical in mouse R2 protein strongly interacts with the diferric center. Removing the tyrosyl radical in the active mouse R2 protein results in irreversible structural changes of the diferric cluster leading to an inactivation of the protein. Therefore the met form of mouse R2 protein can not be stabilized. Unlike mouse R2, the tyrosyl radical of M. tuberculosis R2 protein exhibits extremely weak magnetic interaction with the diferric center. The met form of M. tuberculosis R2 can be easily obtained and stabilized by the treatment of the active enzyme with hydroxyurea. The results of the chemical reduction of the diferric/radical center in the native mouse R2 protein as well as in two mutants (D266A and Y370W) in a proposed electron transfer pathway indicate that in all cases the second order rate constants in the mutants are comparable or faster than in native protein suggesting that the proposed radical transfer pathway is not important for the chemical reduction to proceed.Studying the oxidation of the fully reduced mouse R2 protein by different non-oxygen oxidants, we have demonstrated that the transitions between Fe(III)Fe(III), Fe(II)Fe(II) and Fe(II)Fe(III) states of this protein are fully reversible. The possibility to form a proper binuclear iron center without using molecular oxygen as an oxidant suggests that the _-oxo-bridge in mouse R2 protein does not necessary need molecular oxygen to be formed. Application of the low temperature reduction to mouse and M. tuberculosis R2 proteins demonstrated a presence of two structurally different diferric clusters giving rise to two distinct mixed-valent EPR signals. Whereas the shape of the mixed-valent EPR signal generated by _-irradiation at 77 K in mouse R2 protein is significantly affected by the presence of the tyrosyl radical, we did not observe any effect of the tyrosyl radical presence on the shape of mixed - valent signal generated in M. tuberculosis R2.
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| 2. |
- Persson, Annika, 1964-
(author)
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Essential conserved active site residues in class Ia ribonucleotide reductase from Escherichia coli : identification of radical reaction intermediates and radical transfer intermediates
- 1999
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Doctoral thesis (other academic/artistic)abstract
- The enzyme ribonucleotide reductase catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. The class Ia enzyme from Escherichia coli consists of two homodimeric subunits denoted protein R1 and protein R2, each inactive alone. Protein R1 contains the active site and allosteric effector sites. Protein R2 contains a stable tyrosyl radical and a dinuclear iron-oxo site essential for catalysis. A radical based reaction mechanism and a radical transfer pathway between R1 and R2 has been proposed.The three conserved active site residues Glu441, Asn437 and Ser224 were studied. Their functions in catalysis were adressed with site-directed mutagenesis. The mutant proteins were characterized with biochemical, biophysical and structural methods. A carboxylate at position 441 is the minimum requirement for catalysis. Glu441 contributes to substrate binding. Its role as a general base in the first part of the reaction mechanism and as a general acid in the second part of the reaction mechanism is corroborated. The residues Asn437 and Ser224 are essential for catalysis and participate in the first part of the reaction.E441Q is a suicidal protein converting the normal substrate into a mechanism-based inhibitor. Consecutive reactions with at least three intermediates were identified. The intermediates are free radical species, a cysteine-localized radical, a nucleotide-localized radical and a tyrosyl radical in the radical transfer pathway were identified. This is the first direct evidence for kinetically coupled radical transfer and reaction intermediates in ribonucleotide reductase.
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