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Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-9626-3670
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Lund University, Sweden.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-0265-1873
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-2575-9913
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Number of Authors: 272022 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 11, article id e79226Article in journal (Refereed) Published
Abstract [en]

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

Place, publisher, year, edition, pages
2022. Vol. 11, article id e79226
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-212704DOI: 10.7554/ELIFE.79226ISI: 000932840400001PubMedID: 36083619Scopus ID: 2-s2.0-85138126660OAI: oai:DiVA.org:su-212704DiVA, id: diva2:1718513
Available from: 2022-12-13 Created: 2022-12-13 Last updated: 2024-06-03Bibliographically approved
In thesis
1. High (valent) on O2: Ribonucleotide Reductase and Methane Monooxygenase
Open this publication in new window or tab >>High (valent) on O2: Ribonucleotide Reductase and Methane Monooxygenase
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Macromolecular X-ray crystallography (MX) is a powerful method to investigate protein structures. However, proteins with redox-active centres and radicals are very susceptible to photoreduction. It is therefore challenging to acquire structural details of redox-active centres in defined oxidation states or protein radicals using synchrotron radiation. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) radiation mitigates this problem. XFELs produce intense pulses of femtosecond length that give rise to diffraction before photoinduced movement can occur in the illuminated protein. Additionally, SFX allows experiments at room temperature and induction of reactions in crystallo

In this thesis two different redox-active enzyme systems were investigated with MX and SFX. The first part examines ribonucleotide reductase (RNR). RNR is the only known enzyme to synthesize de novo deoxyribonucleotides, the building blocks of DNA. Class I RNR consists of a small subunit R2 and a large subunit R1. R2 generates a radical in an oxygen dependent way and delivers it to R1 for ribonucleotide reduction. After catalysis the radical is transferred back to R2 until further use. Class I RNR is divided in five subclasses, mostly based on their mechanism of radical generation. In Paper I class Ib R2 is investigated. R2b binds two manganese ions that react with superoxide to produce a radical. The superoxide is provided by a small flavoprotein, NrdI, bound to R2. When exposed to molecular oxygen, reduced NrdI generates superoxide that is transferred to the R2 active site. Here two SFX structures of reduced and oxidized NrdI in complex with R2 are presented and it is suggested how the binding and NrdI oxidation state could influence the superoxide production. In Paper II the SFX structure of a R2e protein radical is presented. Class Ie R2 contains a metal-free active site. The comparison of the radical structure with a ground state structure highlights the changes induced by radical formation. A mechanism for the initiation of the radical transfer to R1 is proposed based on the structural details observed. In Paper III light is shed on a new variant of R2e. Three of the typically conserved active site residues are mutated in R2e; from three glutamates to valine, proline and lysine (VPK) or to glutamine, serine and lysine (QSK). Other publications, including Paper II, describe the VPK mutation but as of now the QSK variant has not been examined. Here, crystal structures of a R2e QSK protein are shown. A tyrosine close to the active site is post-translationally modified to a dihydroxyphenylalanine (DOPA). The amount of modified protein is shown to scale with the coexpression of other proteins of the RNR operon. 

The second redox-active enzyme investigated is soluble methane monooxygenase (sMMO). sMMO oxidizes methane to methanol and is produced by methanotrophs; bacteria that use methane as their sole carbon source. Methane is a potent greenhouse gas and can be found in ever increasing concentrations in the atmosphere due to human activities; sMMO is thus a compelling target for biotechnological development. Paper IV presents SFX structures of the catalytic subunit MMOH in complex with its small regulatory subunit MMOB in the oxidized and reduced resting state. It is also demonstrated that the complex can undergo the catalytic cycle in crystallo, allowing investigation of reaction cycle intermediates in the future. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2023. p. 69
Keywords
Ribonucleotide Reductase, Methane Monooxygenase
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-221059 (URN)978-91-8014-502-2 (ISBN)978-91-8014-503-9 (ISBN)
Public defence
2023-10-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, and online via Zoom, public link is available at the department website, Stockholm, 09:00 (English)
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Supervisors
Available from: 2023-10-04 Created: 2023-09-14 Last updated: 2023-09-27Bibliographically approved

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John, JulianeAurelius, OskarSrinivas, VivekSaura, PatriciaKaila, Ville R. I.Lebrette, HugoHögbom, Martin

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