| Autor: |
Katsyv A; Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main 60438, Germany., Kumar A; Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main 60438, Germany.; SYNMIKRO Research Center and Department of Chemistry, Philipps-University of Marburg, Marburg 35032, Germany., Saura P; Department of Biochemistry and Biophysics, Stockholm University, Stockholm 10691, Sweden., Pöverlein MC; Department of Biochemistry and Biophysics, Stockholm University, Stockholm 10691, Sweden., Freibert SA; Institut für Zytobiologie im Zentrum SYNMIKRO, Philipps-University of Marburg, Marburg 35032, Germany.; Core Facility 'Protein Biochemistry and Spectroscopy', Marburg 35032, Germany., T Stripp S; Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, Berlin 14195, Germany., Jain S; Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main 60438, Germany., Gamiz-Hernandez AP; Department of Biochemistry and Biophysics, Stockholm University, Stockholm 10691, Sweden., Kaila VRI; Department of Biochemistry and Biophysics, Stockholm University, Stockholm 10691, Sweden., Müller V; Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main 60438, Germany., Schuller JM; SYNMIKRO Research Center and Department of Chemistry, Philipps-University of Marburg, Marburg 35032, Germany. |
| Abstrakt: |
Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO 2 , but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H 2 ). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P) + and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P) + binding affinity via reduction of a nearby iron-sulfur cluster, HydABC switches between the exergonic NAD(P) + reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases. |
