Electron Transfer Flavoproteins (ETFs) are electron-transporting proteins that containflavin adenine dinucleotide (FAD) at their active sites. There are two different types of ETF: oneonly performs electron transfer between a physiological electron donor and acceptor while theother has the additional capacity to catalyze electron bifurcation. Electron bifurcation involvestaking a pair of electrons from a median-potential reductant such as NADH and sending theelectrons separately along low- and high-potential pathways in a way that is overallthermodynamically favorable. In this way, it is possible to acquire the low-potential reducingequivalents needed for carbon and nitrogen fixation in many organisms, particularlymethanogenic archaea and acetogenic bacteria. We know that the simple ETFs bind anequivalent of adenosine monophosphate (AMP) in addition to their FAD while the bifurcatingETFs instead have a second equivalent of FAD which is the site of the primary bifurcation event.X-ray crystal structures of both bifurcating and non-bifurcating ETFs are known, but the specificstructural features surrounding the bifurcating FAD that allow binding of the second equivalentof FAD are not understood. We propose to undertake site-directed mutagenesis studies to converta bifurcating ETF to a non-bifurcating one, and vice versa, via using engineered genes. In thisway, we hope to deepen the understanding of how the protein environment of the bifurcatingFAD enables bifurcation.