The energy-transducing NADH-ubiquinone (Q) oxidoreductase of Paracoccus denitrificans is composed of 14 dissimilar subunits and contains at least four iron-sulfur clusters [Yagi, T. (1993) Biochim. Biophys. Acta 1141, 1-17]. The complete DNA sequence of the gene cluster encoding the energy-transducing NADH-Q oxidoreductase of P. denitrificans has been determined. This paper reports the expression of the 25-kilodalton (kDa) (NQO2) subunit of the P. denitrificans enzyme complex in Escherichia coli and the characterization of the iron-sulfur cluster bound to the expressed subunit. The 25-kDa subunit was expressed in the cytoplasmic phase but not in the membrane fraction of E. coli cells and then purified using an affinity nickel chelation column. The purified subunit contains 1.44 mol of non-heme iron and 1.33 mol of acid-labile sulfide/mol of subunit. EPR analysis of the reduced form of this subunit indicates that the expressed subunit contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with g values of gx,y,z = 1.913, 1.942, and 1.996, which is very similar to the spectrum of the [2Fe-2S] cluster in the resolved flavoprotein II subfraction (subunit 24 + 9 kDa) of bovine heart complex I [Ragan, C. I., Galante, Y. M., Hatefi, Y., & Ohnishi, T. (1982) Biochemistry 21, 590-594; Ohnishi, T., Ragan, C. I., & Hatefi, Y. (1985) J. Biol. Chem. 260, 2782-2788]. The assignment of the binuclear iron-sulfur cluster of the 25-kDa subunit to an EPR-visible iron-sulfur cluster in the Paracoccus NADH-Q oxidoreductase in situ is discussed.