The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 unlike subunits and contains one FMN and at least five EPR-detectable iron-sulfur clusters. The 14 subunits are designated NQO1 through NQO14. The expression and partial characterization of the NQO4, -5, and -6 subunits have been performed. The NQO4, -5, and -6 subunits were individually expressed in Escherichia coli. The NQO4 subunit was expressed in both the cytoplasmic phase and membrane fraction, the NQO5 subunit in the cytoplasmic phase only, and the NQO6 subunit in the membrane fraction only. The NQO4 and NQO5 subunits were purified from cytoplasmic phase. Neither subunit contains non-heme iron or acid-labile sulfide, suggesting that the NQO4 or NQO5 subunit is not an iron-sulfur subunit. The antibodies against the NQO4, -5, and -6 subunits cross-reacted with their counterpart subunits in bovine heart complex I. The NQO4, -5, and -6 subunits in membrane-bound P. denitrificans NDH-1 were extracted by treatment at alkaline pH ( > or = 10) or with chaotropes (NaBr, Nal, and urea), suggesting that these subunits are localized in the peripheral part (not in the membrane sector) of the enzyme complex similar to the NQO1, -2, and -3 subunits. In addition, the subunit stoichiometry of NQO1 through -6 of the membrane-bound P. denitrificans NDH-1 has been determined by radioimmunoassays. There is 1 mol each of the NQO1 through -6 subunits per mol of the P. denitrificans NDH-1.