ABSTRACT
The proton-translocating reduced nicotinamide adenine dinucleotide- (NADH-) quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 different subunits (NQO1-14). In addition, this enzyme complex houses one flavin mononucleotide (FMN) and 7-8 iron-sulfur clusters as cofactors. The expression and partial characterization of the NQO7 subunit, one of the seven subunits that constitute the hydrophobic sector of the enzyme complex, have been performed and are reported here. Expression of the NQO7 subunit was achieved by use of the glutathione-S-transferase (GST) fusion system together with Escherichia coli strains BLR(DE3)pLysS and BL21(DE3)pLysS. The GST-fused NQO7 subunit was expressed in the membrane fraction of the host cells and was extracted from the membranes by nonionic detergents (Triton X-100, dodecyl maltoside). The extracted polypeptide was purified by glutathione affinity column chromatography and characterized. The isolated GST-fused NQO7 subunit (but not the GST alone) was determined to interact with phospholipid vesicles and suppress the membrane fluidity. Antibodies against both the N- and C-terminal regions of the deduced primary structure of the NQO7 subunit reacted with a single band (15 kDa) of the Paracoccus membranes. By use of immunochemical and cysteine residue modification techniques, the topology of the Paracoccus NQO7 subunit in the membranes has been examined. The data suggest that the Paracoccus NQO7 subunit contains three transmembrane segments and that its N- and C-terminal regions are directed toward the cytoplasmic and periplasmic phases of the membrane, respectively. The proposed topology of the GST-fused NQO7 subunit expressed in E. coli membranes is consistent with that of the NQO7 subunit in the Paracoccus membranes.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , NAD/chemistry , Paracoccus denitrificans/enzymology , Peptide Fragments/chemistry , Quinone Reductases/chemistry , Amino Acid Sequence , Animals , Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/isolation & purification , Cattle , Cell Membrane/enzymology , Escherichia coli/enzymology , Escherichia coli/genetics , Glutathione Transferase/genetics , Humans , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sequence Homology, Amino AcidABSTRACT
In beef heart mitochondria it has been found that the Km for coenzyme Q10 of the NADH oxidation system is in the range of the membrane concentration of the quinone; this is contrary to succinate oxidation which is in Vmax with respect to quinone content. The same proportional difference between the two systems is maintained in their affinities for the exogenous acceptor CoQ1 in non-extracted mitochondria. The Km of succinate- coenzyme Q reductase for CoQ1 is reversibly lowered in CoQ-depleted mitochondria; while in contrast the Km for NADH-coenzyme Q reductase is reversibly increased by CoQ extraction. Incorporation of exogenous quinones by co-sonication with submitochondrial particles, as evidenced by fluorescence quenching of pyrene, enhances NADH-cytochrome c reductase activity in accordance with the lack of saturation of the former system.
