EXPRESSION AND CHARACTERIZATION OF THE 66-KILODALTON (NQO3) IRON-SULFUR SUBUNIT OF THE PROTON-TRANSLOCATING NADH-QUINONE OXIDOREDUCTASE OF PARACOCCUS-DENITRIFICANS

The proton translocating NADH-quinone oxidoreductase (NDH-1) of Paraco ccus denitrificans is composed of at least 14 dissimilar subunits whic h are designated NQO1-14 and contains one noncovalently bound FMN and at least five EPR-visible iron-sulfur clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of the deduced primary structures of the subunits with consensus sequences for the cofactor binding sit es has predicted that NQO1, NQO2, NQO3, NQO9, and probably NQO6 subuni ts are cofactor binding subunits. Previously, we have reported that th e NQO2 (25 kDa) subunit was overexpressed as a water-soluble protein i n Escherichia coli and was found to ligate a single [2Fe-2S] cluster w ith rhombic symmetry (g(x,y,z) = 1.92, 1.95, and 2.00) (Yano, T., Sled ', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In the present study, the NQO3 (66 kDa) subunit, which is equivalent t o the 75-kDa subunit of bovine heart Complex I, was overexpressed in E . coli. The expressed NQO3 subunit was found predominantly in the cyto plasmic phase and was purified by ammonium sulfate fractionation and a nion-exchange chromatography. The chemical analyses and W-visible and EPR spectroscopic studies showed that the expressed NQO3 subunit conta ins at least two distinct iron-sulfur clusters: a [2Fe-2S] cluster wit h axial EPR signals (g(perpendicular to,parallel to) = 1.934 and 2.026 , and L(perpendicular to,parallel to) = 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster with rhombic symmetry (g(x,y,z) = 1.892, 1.928, and 2 .063, and L(x,y,z) = 2.40, 1.55, and 1.75 millitesla). The midpoint re dox potentials of [2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are -472 an d -391 mV, respectively. The tetranuclear cluster in the isolated NQO3 subunit is sensitive toward oxidants and converts into [3Fe-4S] form. The assignment of these iron-sulfur clusters to those identified in t he P. denitrificans NDH-1 enzyme complex and the possible functional r ole of the NQO3 subunit is discussed.