Hydrogen is an attractive clean fuel that can be produced biologically either from water or by fermentation of organic compounds. Although organic acids are completely fermented to H₂ and CO₂ by photosynthetic organisms, need for light in this process makes this uneconomical for commercial production of H₂. Fermentation of sugars to hydrogen is fraught with lower yields that are less than the theoretical maximum of 4 H₂ per glucose. To increase the H₂ yield, we are developing a new pathway that couples NADH oxidation to H₂ production. NADH-ferredoxin oxidoreductase (NFOR) from the hydrogenosome of an anaerobic protozoan was cloned and expressed in Escherichia coli as the first enzyme of this pathway. The recombinant enzyme was purified as a heterodimer (46,600 and 25,200 Da). The holoenzyme had a [2Fe-2S] cluster that was localized in the small subunit. Low temperature EPR spectra of dithionite reduced active enzyme confirmed the presence of the [2Fe-2S] cluster with a g_xyz of 1.92, 1.95 and 2.008. Kinetic properties of the enzyme are comparable with the enzyme purified from hydrogenosomes. The enzyme reduced methyl viologen, benzyl viologen and Clostridium pasteurianum ferredoxin, electron acceptors with a mid-point redox potential that are more negative than NADH/NAD+ (-320 mV) using NADH as the electron donor. The ability to reduce methyl viologen or ferredoxin suggests that the enzyme can couple NADH oxidation to hydrogenase and H₂ production with methyl viologen or ferredoxin as intermediate electron carrier towards increasing fermentative H₂ yield from glucose to higher than the theoretical maximum of 4.0.