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To metal, or not to metal: Diverse mechanisms of O2-activation and radical storage in the ferritin superfamily
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-0265-1873
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins in the Ferritin-like superfamily are characterized by a four alpha-helical structural motif. These proteins are distributed across all three kingdoms of life and perform a wide range of functions. Several members in this protein superfamily can activate dioxygen using a di-metal active site coordinated by four carboxylate and two histidine amino acid residues. The resulting diverse set of dioxygen activated intermediates is used in nature to perform complex redox chemical reaction in cells. The R2 subunit of class I Ribonucleotide reductase and soluble Methane monooxygenase are the most well-characterized groups of proteins in this superfamily. Upon oxygen (or reduced-oxygen) activation of the di-metal site, the R2 subunit can generate a catalytic radical required for the conversion of ribonucleotides to deoxyribonucleotides, while soluble Methane monooxygenase can oxidize methane to methanol in an alternative form of carbon assimilation.

The work presented in this thesis aims to better understand metal selectivity, working and the regulation of substrate specificity in various proteins of the Ferritin-like superfamily, and the development of a novel method to study radiation-sensitive intermediates. The papers discussed in this thesis present crystallographic and spectroscopic studies of several Ferritin-like superfamily proteins.

In paper I, the assembly mechanisms of the heterodinuclear manganese-iron cofactor in a class Ic R2 protein and an R2-like ligand-binding oxidase are compared. Paper II presents the discovery of a novel radical-generating subunit subclass of Ribonucleotide reductase in Mollicutes, including mycoplasma pathogens, that breaks the paradigm of metal requirement for radical translocation and catalysis. This new subclass, denoted class Ie, is shown to instead use an unprecedented modified tyrosine DOPA residue in its four-helix bundle for radical translocation and storage. Paper III presents a new X-ray free-electron laser sample delivery system that combines acoustic droplet ejection with a drop-on-tape setup, allowing simultaneous multimodal X-ray diffraction and X-ray emission data collection. This setup is also shown to support photochemical and chemical activation of catalysis in crystals, allowing the study of radiation-sensitive transient reaction intermediates. We used this setup in paper IV to solve the first radiation damage-free crystallographic structures of the soluble methane monooxygenase hydroxylase and its regulatory subunit complex from Methylosinus trichosporium OB3b. The high-resolution crystal structures of the complex, in both di-ferrous and di-ferric oxidation states, illustrate the structural reorganization in the hydroxylase subunit upon binding to the regulatory subunit.

These results illustrate the functional range and flexibility in the Ferritin-like protein superfamily. Including the distinctive metal discrimination in heterodinuclear metalloproteins, influencing substrate specificity in sMMO, and using a novel metal-free DOPA radical to catalyze ribonucleotide reduction in the class Ie R2 subclass. Experiments using the novel ADE-DOT setup also showed promising progress towards determining the highly sought-after structures of di-metal oxygen activated intermediates such as X and Q in subclass Ia R2 and sMMO, respectively.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 84
Keywords [en]
Metalloprotein, di-metal, Oxygen activation, Substrate oxidation, Ribonucleotide reductase, Radical, DOPA, ADE-DOT, X-Ray free-electron laser, soluble Methane monooxygenase
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-174024ISBN: 978-91-7797-887-9 (print)ISBN: 978-91-7797-888-6 (electronic)OAI: oai:DiVA.org:su-174024DiVA, id: diva2:1356920
Public defence
2019-11-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius Väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2019-10-23 Created: 2019-10-02 Last updated: 2019-10-11Bibliographically approved
List of papers
1. Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins
Open this publication in new window or tab >>Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins
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2016 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 162, p. 164-177Article in journal (Refereed) Published
Abstract [en]

A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of dass Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is undear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn-II and Fe-II. Using EPR and Mfissbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn-II promotes heterobimetallic cofactor assembly. In the absence of Fe-II, R2c cooperatively binds Mn-II at both metal sites, whereas R2lox does not readily bind Mn-II at either site. Heterometallic cofactor assembly is favored at substoichiometric Feu concentrations in R2lox. Fe-II and Mn-II likely bind to the protein in a stepwise fashion, with Feu binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn-II by Fe-II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn-II/Fe-II concentrations in the host organisms from which they were isolated.

Keywords
Ferritin, Di-metal carboxylate protein, Ribonucleotide reductase, EPR spectroscopy, Mossbauer spectroscopy, X-ray crystallography
National Category
Biological Sciences Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-136945 (URN)10.1016/j.jinorgbio.2016.04.019 (DOI)000388059600019 ()27138102 (PubMedID)
Available from: 2016-12-23 Created: 2016-12-19 Last updated: 2019-12-04Bibliographically approved
2. Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens
Open this publication in new window or tab >>Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens
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2018 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 563, p. 416-420Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5,6,7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-161580 (URN)10.1038/s41586-018-0653-6 (DOI)000450048400063 ()
Available from: 2018-11-01 Created: 2018-11-01 Last updated: 2019-12-04Bibliographically approved
3. Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers
Open this publication in new window or tab >>Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers
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2017 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 14, no 4, p. 443-449Article in journal (Refereed) Published
Abstract [en]

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem IIII, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-142628 (URN)10.1038/nmeth.4195 (DOI)000397900500029 ()28250468 (PubMedID)
Available from: 2017-05-17 Created: 2017-05-17 Last updated: 2019-12-04Bibliographically approved
4. High Resolution XFEL Structure of the Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States
Open this publication in new window or tab >>High Resolution XFEL Structure of the Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-174017 (URN)
Available from: 2019-10-02 Created: 2019-10-02 Last updated: 2019-10-02Bibliographically approved

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