Abstract
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.
Highlights
The respiratory chain of aerobically grown Paracoccus denitrificans consists of NADH-quinone oxidoreductase,1 succinate-quinone oxidoreductase
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
By expressing and characterizing NQO2 altered by site-directed mutagenesis, we have shown that the [2Fe–2S] cluster is coordinated by four conserved cysteine resframe; Complex I, mitochondrial proton-translocating NADH-ubiquinone oxidoreductase; FP, flavoprotein; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol
Summary
Construction of Expression Vectors for NQO1 and NQO2 Subunits—A 1.5-kilobase pair BsmAI fragment containing the full-length NQO1 gene was digested from pXT-1 [10], blunt-ended with a DNA polymerase Klenow fragment, and ligated into SmaI-digested cloning vector pTZ19U. The clarified supernatant was applied to a Sephadex G-50 gel filtration column (1.0 ϫ 30 cm) equilibrated with 50 mM Tris-HCl buffer (pH 8.5) containing 5.0 mM DTT in order to remove excess FMN, iron, and sulfide. Gel Filtration Analysis—The purified reconstituted FP subcomplex (1.0 mg) was applied to a gel filtration column (Bio-Rad A-5m, 1.0 ϫ 45 cm), equilibrated with 50 mM Tris-HCl buffer (pH 8.5), containing 5 mM DTT and 300 mM NaCl, and eluted at a flow rate of 0.5 ml/min. S. Biochemical Corp.; [␣-35S]thio-dATP was from Amersham Corp.; NADH, NADϩ, NADPH, DTT, and alkaline phosphatase-conjugating affinity-purified antibodies to rabbit IgG were from Calbiochem; deamino-NADH, FMN, FAD, and riboflavin were purchased from Sigma; expression vectors, pET11a, pET16b, E. coli strain BL21(DE3)pLysS, and the nickel chelation resin were from Novagen; carbenicillin was from ICN.
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