This study reports the expression of the flavoprotein (FP) subcomplex of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from Paracoccus denitrificans, which is composed of the NQO1 (50 kDa) and the NQO2 (25 kDa) subunits. The two subunits are co-expressed in Escherichia coli using a double expression plasmid system. The expressed subunits form a water-soluble heterodimer complex with 1:1 stoichiometry. The expressed complex contained one [2Fe 2S] cluster but almost no FMN or [4Fe 4S] cluster. The two latter prosthetic groups could be partially reconstituted with FMN, Na2S, and (NH4)2Fe(SO4)2 in vitro under anaerobic conditions. The reconstituted FP subcomplex showed EPR signals from two distinct species of iron-sulfur cluster. One resonance transition originates from a [2Fe-2S] cluster with g values of gx,y,z = 1.92, 1.95, and 2.00 and slow spin relaxation, which was tentatively assigned to the cluster N1a. These EPR properties are very similar to those reported for the NQO2 subunit expressed alone (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). The other originates from a [4Fe 4S] cluster with g values of gx,y, z = 1.87, 1.94, and 2.04 and fast relaxing behavior, which are reminiscent of the cluster N3 in the membrane bound enzyme complex. After reconstitution with FMN, the FP subcomplex catalyzed electron transfer from NADH and from deamino-NADH to a variety of electron acceptors. The enzymatic properties of the FP subcomplex, reconstituted with FMN and iron-sulfur, correspond to those of the isolated P. denitrificans NADH-dehydrogenase complex.