Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]- Hydrogenase Complex HydABC

Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO2, 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 (H2). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experi-ments, 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.

Automated summary

Electron bifurcation is a fundamental energy coupling mechanism used by microorganisms in anoxic conditions to reduce CO2 using hydrogen. The enzyme responsible for these reactions, HydABC, uses a single flavin mononucleotide (FMN) cofactor to transfer electrons to NAD(P)+ and low-potential ferredoxins (Fd), and switches between NAD(P)+ reduction and Fd reduction modes. Understanding the mechanistic principles of electron-bifurcating hydrogenases can provide insight into energy conversion processes in microorganisms.