Trichomonas vaginalis generates reduced ferredoxin within a unique subcellular organelle, hydrogenosome that is used as a reductant for H-2 production. Pyruvate ferredoxin oxidoreductase and NADH dehydrogenase (NADH-DH) are the two enzymes catalyzing the production of reduced ferredoxin. The genes encoding the two subunits of NADH-DH were cloned and expressed in Escherichia coli. Kinetic properties of the recombinant heterodimer were similar to that of the native enzyme from the hydrogenosome. The recombinant holoenzyme contained 2.15 non-heme iron and 1.95 acid-labile sulfur atoms per heterodimer. The EPR spectrum of the dithionite-reduced protein revealed a [2Fe-2S] cluster with a rhombic symmetry of g(xyz)=1.917, 1.951, and 2.009 corresponding to cluster N1a of the respiratory complex I. Based on the Fe content, absorption spectrum, and the EPR spectrum of the purified small subunit, the [2Fe-2S] cluster was located in the small subunit of the holoenzyme. This recombinant NADH-DH oxidized NADH and reduced low redox potential electron carriers, such as viologen dyes as well as Clostridium ferredoxin that can couple to hydrogenase for H-2 production from NADH. These results show that this unique hydrogenosome NADH dehydrogenase with a critical role in H-2 evolution in the hydrogenosome can be produced with near-native properties in E. coli for metabolic engineering of the bacterium towards developing a dark fermentation process for conversion of biomass-derived sugars to H-2 as an energy source.