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Manganese and Iron Heterodimers and Homodimers in Enzymes: Insights from Density Functional Theory
Stockholm University, Faculty of Science, Department of Physics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The enzyme ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides, the building blocks of DNA, and is essential for all organisms. Canonical class I RNR R2 proteins use a diiron cofactor to generate a tyrosyl radical, which is required for catalysis. Recent discoveries have established that the different subgroups of class I RNR employ different metal cofactors. Class Ib R2 (R2F) utilizes a dimanganese cofactor and a flavoprotein to generate the tyrosyl radical. Class Ic R2 (R2c) lacks the radical-bearing tyrosine, and instead has an oxidized heterodinuclear manganese-iron center, the first known redox active MnFe cofactor. A second group of MnFe proteins with different functions, denoted R2-like ligand binding oxidases (R2lox), was later identified. R2lox proteins are capable of performing two-electron oxidations and are believed to be hydrocarbon oxidases. In the present thesis density functional theory, a quantum mechanical method, is employed to study the manganese and iron heterodimers and homodimers in the R2 and R2lox proteins, with the aim to shed light on the mechanistic details and stress the main features of the alternative metal centers. Some of the questions addressed are the radical generation with the homodimers and heterodimer in R2, the radical transfer between R2 and the RNR catalytic subunit, and the function of R2lox. A Mn(IV)Fe(III) state is shown to be an equally strong oxidant as a tyrosyl radical, giving a rationalization for the presence of the heterodimer in R2c. A reaction mechanism for the formation of an unprecedented tyrosine-valine crosslink catalyzed by the heterodimer in R2lox is modeled, and the potential of the protein to perform hydroxylations of hydrocarbons based on calculated barriers for methane hydroxylation is discussed. An energetically possible reaction mechanism is suggested for activation of dimanganese R2F by hydrogen peroxide, and a hypothetical role of the flavoprotein in radical generation is proposed.

Place, publisher, year, edition, pages
Stockholm: Department of Physics, Stockholm University , 2012. , 86 p.
Keyword [en]
Ribonucleotide reductase, manganese, iron, density functional theory
National Category
Theoretical Chemistry Biochemistry and Molecular Biology Inorganic Chemistry
Research subject
Chemical Physics
Identifiers
URN: urn:nbn:se:su:diva-78814ISBN: 978-91-7447-543-2 (print)OAI: oai:DiVA.org:su-78814DiVA: diva2:544181
Public defence
2012-09-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript.

Available from: 2012-08-23 Created: 2012-08-13 Last updated: 2015-10-27Bibliographically approved
List of papers
1. Density Functional Theory Study of the Manganese-Containing Ribonucleotide Reductase from Chlamydia trachomatis: Why Manganese Is Needed in the Active Complex
Open this publication in new window or tab >>Density Functional Theory Study of the Manganese-Containing Ribonucleotide Reductase from Chlamydia trachomatis: Why Manganese Is Needed in the Active Complex
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 9, 1878-1887 p.Article in journal (Refereed) Published
Abstract [en]

The active center of Chlamydia trachomatis (Ct) ribonucleotide reductase (RNR) has been studied using B3LYP hybrid density functional theory. Class Ic Ct RNR lacks the radical-bearing tyrosine that is crucial for activity in conventional class I (subclass a and b) RNR. Instead of the Fe(III)Fe(III)Tyr(rad) active state in conventional class I, Ct RNR has Mn(IV)Fe(III) at the metal center of subunit H. Based on calculated (H+, e(-))-binding energies for Ct R2, iron-substituted Ct R2, and R2 from Escherichia coli (Ec), an explanation is proposed for why the enzyme needs this novel metal center. Mn(IV) is shown to be an equally strong oxidant as the tyrosyl radical in Ec R2. Fe(IV), however, is a much too strong oxidant and would therefore not be possible in the active cofactor. The structure of the catalytic center of the active state, such as protonation state and position of Mn, is discussed. Ct R2 has a different ligand structure than conventional class I R2 with a fourth Glu (like MMO) instead of three Glu and one Asp. Calculations indicate that, in the presence of Tyr, Glu at this position is less flexible than Asp, whereas with Phe both Glu and Asp are equally flexible. This may be a reason why conventional class I RNR has an Asp, while Ct R2, lacking the tyrosine, has a Glu.

Keyword
correlated molecular calculations, continuum dielectric theory, gaussian-basis sets, intermediate-x, protein r2, electron-transfer, cofactor, activation, hydrogen, cluster
National Category
Physical Sciences
Research subject
Chemical Physics
Identifiers
urn:nbn:se:su:diva-60370 (URN)10.1021/bi801695d (DOI)000263896100005 ()
Note
authorCount :2Available from: 2011-08-19 Created: 2011-08-16 Last updated: 2017-12-08Bibliographically approved
2. Oxygen cleavage with manganese and iron in ribonucleotide reductase from Chlamydia trachomatis
Open this publication in new window or tab >>Oxygen cleavage with manganese and iron in ribonucleotide reductase from Chlamydia trachomatis
2011 (English)In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 16, no 4, 553-565 p.Article in journal (Refereed) Published
Abstract [en]

The oxygen cleavage in Chlamydia trachomatis ribonucleotide reductase (RNR) has been studied using B3LYP* hybrid density functional theory. Class Ic C. trachomatis RNR lacks the radical-bearing tyrosine, crucial for activity in conventional class I (subclass a and b) RNR. Instead of the Fe(III)Fe(III)-Tyr(rad) active state, C. trachomatis RNR has a mixed Mn(IV)Fe(III) metal center in subunit II (R2). A mixed MnFe metal center has never been observed as a radical cofactor before. The active state is generated by reductive oxygen cleavage at the metal site. On the basis of calculated barriers for oxygen cleavage in C. trachomatis R2 and R2 from Escherichia coli with a diiron, a mixed manganese iron, and a dimanganese center, conclusions can be drawn about the effect of changing metals in R2. The oxygen cleavage is found to be governed by two factors: the redox potentials of the metals and the relative stability of the different peroxides. Mn(IV) has higher stability than Fe(IV), and the barrier is therefore lower with a mixed metal center than with a diiron center. With a dimanganese center, an asymmetric peroxide is more stable than the symmetric peroxide, and the barrier therefore becomes too high. Calculated proton-coupled redox potentials are compared to identify three possible R2 active states, the Fe(III)-Fe(III)-Tyr(rad) state, the Mn(IV)Fe(III) state, and. the Mn(IV)Mn(IV) state. A tentative energy profile of the thermodynamics of the radical transfer from R2 to subunit I is constructed to illustrate how the stability of the active states can be understood from a thermodynamical point of view.

Keyword
Ribonucleotide reductase, Oxygen cleavage, Manganese, Iron, Density functional theory
National Category
Physical Sciences
Research subject
Chemical Physics
Identifiers
urn:nbn:se:su:diva-68806 (URN)10.1007/s00775-011-0755-1 (DOI)000290284600003 ()
Note
authorCount :2Available from: 2012-01-09 Created: 2012-01-07 Last updated: 2017-12-08Bibliographically approved
3. A comparison of two-electron chemistry performed by the manganese and iron heterodimer and homodimers
Open this publication in new window or tab >>A comparison of two-electron chemistry performed by the manganese and iron heterodimer and homodimers
2012 (English)In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 17, no 3, 363-373 p.Article in journal (Refereed) Published
Abstract [en]

Two-electron chemistry with an iron dimer, a manganese dimer, and a manganese-iron dimer as a catalyst has been modeled using B3LYP* hybrid density functional theory. The recently discovered MnFe proteins form (at least) two functionally distinct groups, performing radical generation (class Ic ribonucleotide reductase subunit II) and substrate oxidations (subunit II-like ligand-binding oxidases, R2lox), respectively. Proteins from the latter group appear to be functionally similar to the diiron carboxylate proteins that perform two-electron oxidations of substrates, such as methane monooxygenase. To qualitatively determine the potential role of a MnFe center in R2lox, methane hydroxylation with the MnFe heterodimer and with the FeFe and MnMn homodimers is studied. The redox potential of the active state of the Mn(IV)Fe(IV) heterodimer is about 7 kcal mol(-1) lower than that of the active state of the Fe(IV)Fe(IV) homodimer, leading to a high barrier for the rate-limiting hydrogen abstraction with the MnFe site. If the entropy loss is not included, the barriers are lower, and the MnFe heterodimer can therefore have a role in R2lox as an oxidase for larger substrates exergonically bound to the protein. A MnMn center has a high barrier both with and without entropy loss. The higher stability of Fe(IV) in the presence of Mn(IV) in the other site compared with a second Fe(IV) suggests an explanation for the presence of the MnFe site in R2lox: to provide a metal center that is capable of two-electron chemistry, and which is more stable and less sensitive to external reductants than an Fe(IV)Fe(IV) site.

Keyword
Manganese, Iron, Heterodinuclear, Metal specificity, Redox tuning
National Category
Physical Sciences
Research subject
Chemical Physics
Identifiers
urn:nbn:se:su:diva-76255 (URN)10.1007/s00775-011-0858-8 (DOI)000301186000004 ()
Note
2Available from: 2012-05-28 Created: 2012-05-10 Last updated: 2017-12-07Bibliographically approved
4. Activation of Dimanganese Class Ib Ribonucleotide Reductase by Hydrogen Peroxide: Mechanistic Insights from Density Functional Theory
Open this publication in new window or tab >>Activation of Dimanganese Class Ib Ribonucleotide Reductase by Hydrogen Peroxide: Mechanistic Insights from Density Functional Theory
2013 (English)In: Inorganic Chemistry, ISSN 0020-1669, E-ISSN 1520-510X, Vol. 52, no 8, 4173-4184 p.Article in journal (Refereed) Published
Abstract [en]

Activation of manganese-dependent class Ib ribonucleotide reductase by hydrogen peroxide was modeled using B3LYP* hybrid density functional theory. Class Ib ribonucleotide reductase R2 subunit (R2F) does not react with molecular oxygen. Instead R2F is proposed to react with H2O2 or HO2-, provided by the unusual flavodoxin protein Nrdl, to generate the observed manganese(III) manganese(III) tyrosyl-radical state. On the basis of the calculations, an energetically feasible reaction mechanism is suggested for activation by H2O2, which proceeds through two reductive half-reactions. In the first reductive half-reaction, H2O2 is cleaved with a barrier of 13.1 kcal mol(-1) [Mn(II)Mn(II) -> Mn(III)Mn(III)], and in the second reductive half-reaction, H2O2 is cleaved with a barrier of 17.0 kcal mol(-1) [Mn(III)Mn(III) -> Mn(IV)Mn(IV)]. Tyrosyl-radical formation from both the Mn(IV)Mn(IV) state and a Mn(III)Mn(IV) state, where an electron and proton have been taken up, is both kinetically and thermodynamically accessible. Hence, chemically, H2O2 is a possible oxidant for the manganese-dependent R2F. The selectivity between the second reductive half-reaction and a competing oxidative reaction, as in manganese catalase, may be the time scale for the availability of H2O2. The role of Nrdl may be to provide H2O2 on the correct time scale.

National Category
Chemical Sciences
Research subject
Chemical Physics
Identifiers
urn:nbn:se:su:diva-90370 (URN)10.1021/ic3008427 (DOI)000317712000015 ()
Note

AuthorCount:2;

Available from: 2013-06-12 Created: 2013-06-03 Last updated: 2017-12-06Bibliographically approved
5. Formation of the Unusual Tyrosine-Valine Crosslink in the Manganese-Iron Heterodimer Oxidase from Mycobacterium tuberculosis
Open this publication in new window or tab >>Formation of the Unusual Tyrosine-Valine Crosslink in the Manganese-Iron Heterodimer Oxidase from Mycobacterium tuberculosis
(English)Manuscript (preprint) (Other academic)
Keyword
Manganese-iron, post-translational crosslink, tyrosine, valine, density functional theory
National Category
Theoretical Chemistry Inorganic Chemistry Biochemistry and Molecular Biology
Research subject
Chemical Physics
Identifiers
urn:nbn:se:su:diva-78813 (URN)
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2012-08-14Bibliographically approved

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