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Isolation of an H2-dependent electron-bifurcating CO2-reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis

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Nomura,  Shunsuke
Department-Independent Research Group Microbial Protein Structure, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Paczia,  Nicole       
Core Facility Metabolomics and small Molecules Mass Spectrometry, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Kahnt,  Jörg
Core Facility Mass Spectrometry and Proteomics, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Shima,  Seigo       
Department-Independent Research Group Microbial Protein Structure, Max Planck Institute for Terrestrial Microbiology, Max Planck Society;

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Citation

Nomura, S., Paczia, N., Kahnt, J., & Shima, S. (2024). Isolation of an H2-dependent electron-bifurcating CO2-reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis. The FEBS Journal. doi:10.1111/febs.17115.


Cite as: https://hdl.handle.net/21.11116/0000-000F-03B7-F
Abstract
In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2. Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin-based electron-bifurcating mechanism. [NiFe]-hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420, respectively. Recently, an Fdh-Hdr complex was purified as a 3-MDa megacomplex that contained Fmd, and its three-dimensional structure was elucidated by cryo-electron microscopy. In contrast, the Mvh-Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1-MDa Mvh-Hdr-Fmd megacomplex from Methanothermobacter?marburgensis. After anion-exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1- and 0.5-MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1-MDa megacomplex was determined to be a dimeric Mvh-Hdr-Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe-4S]-clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh-Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh-Hdr and Fmd in the Mvh-Hdr-Fmd complex. The purified Mvh-Hdr-Fmd megacomplex catalyzed electron-bifurcating reduction of [13C]-CO2 to form [13C]-formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh-Hdr-Fmd megacomplex are electronically connected for the reduction of CO2, which likely involves MvhB polyferredoxin as an electron relay.