The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structure

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with [32P]NADH [Yagi, T., & Dinh, T.M. (1990) Biochemistry 29, 5515-5520]. Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction. This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P. denitrificans. The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16. When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster.     

Characterization of the membrane domain Nqo11 subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans consists of at least 14 unlike subunits (designated Nqo1-14). The NDH-1 is composed of two segments (the peripheral and membrane segments). The membrane domain segment appears to be made up of seven subunits (Nqo7, -8, -10-14). In this report, the characterization of the Paracoccus Nqo11 subunit has been investigated. An antibody against the C-terminal 12 amino acid residues of the Paracoccus Nqo11 subunit (Nqo11c) has been raised. The Nqo11c antibody reacted with a single band (11 kDa) of the Paracoccus membranes and cross-reacted with Rhodobactor capsulatus membranes. The Nqo11 subunit was not able to be extracted from the Paracoccus membranes by NaI or alkaline treatment, unlike the peripheral subunits (Nqo1 and Nqo6). The C-terminal region of the Paracoccus Nqo11 is exposed to the cytoplasmic phase. For further characterization of the Paracoccus Nqo11 subunit, the subunit was overexpressed in Escherichia coli by using the maltose-binding protein (MBP) fusion system. The MBP-fused Nqo11 subunit was expressed in the E. coli membranes (but not in soluble phase) and was extracted by Triton X-100. The isolated MBP-fused Nqo11 subunit interacted with the phospholipid vesicles and suppressed their membrane fluidity. Topological studies of the Nqo11 subunit expressed in E. coli membranes have been performed by using cysteine mapping and immunochemical analyses. The data suggest that the Nqo11 subunit has three transmembrane segments and its C-terminus protrudes into the cytoplasmic phase.     

Characterization and topology of the membrane domain Nqo10 subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (Nqo1-Nqo14). Of these, seven subunits (Nqo7, Nqo8, and Nqo10-14) which are equivalent to the mitochondrial DNA-encoded subunits of complex I constitute the membrane segment of the enzyme complex; the remaining subunits make up the peripheral part of the enzyme. We report here on the biochemical characterization and heterologus expression of the Nqo10 subunit. The Nqo10 subunit could not be extracted from the Paracoccus membranes by NaI or alkaline treatment, which is consistent with the presumed membrane localization. By using the maltose-binding protein (MBP) fusion system, the Nqo10 subunit was overexpressed in Escherichia coli. The MBP-fused Nqo10 was expressed in membrane fractions of the host cell and was extractable by Triton X-100. The extracted fusion protein was then isolated by one-step affinity purification through an amylose column. By using immunochemical methods in conjunction with cysteine-scanning mutagenesis and chemical modification techniques, the topology of the Nqo10 subunit expressed in E. coli membranes was determined. The data indicate that the Nqo10 subunit consists of five transmembrane segments with the N- and C-terminal regions facing the periplasmic and cytoplasmic sides of the membrane, respectively. In addition, the data also suggest that the proposed topology of the MBP-fused Nqo10 subunit expressed in E. coli membranes is consistent with that of the Nqo10 subunit in the native Paracoccus membranes. From the experimentally determined topology together with computer prediction programs, a topological model for the Nqo10 subunit is proposed.     

Characteristics of the energy-transducing NADH-quinone oxidoreductase ofParacoccus denitrificans as revealed by biochemical,biophysical, and molecular biological approaches

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.     

Structural features of the 66-kDa subunit of the energy-transducing NADH-ubiquinone oxidoreductase (NDH-1) of Paracoccus denitrificans.

The structural gene of the Paracoccus denitrificans NADH-ubiquinone oxidoreductase encoding a homologue of the 75-kDa subunit of bovine complex I (NQO3) has been located and sequenced. It is located approximately 1 kbp downstream of the gene coding for the NADH-binding subunit (NQO1) [Xu, X., Matsuno-Yagi, A., and Yagi, T. (1991) Biochemistry 30, 6422-6428] and is composed of 2019 base pairs and codes for 673 amino acid residues with a calculated molecular weight of 73,159. The M(r) 66,000 polypeptide of the isolated Paracoccus NADH dehydrogenase complex is assigned the NQO3 designation on the basis of N-terminal protein sequence analysis, amino acid analysis, and immuno-cross-reactivity. The encoded protein contains a putative tetranuclear iron-sulfur cluster (probably cluster N4) and possibly a binuclear iron-sulfur cluster. An unidentified reading frame (URF3) which is composed of 396 base pairs and possibly codes for 132 amino acid residues was found between the NQO1 and NQO3 genes. When partial DNA sequencing of the regions downstream of the NQO3 gene was performed, sequences homologous to the mitochondrial ND-1, ND-5, and ND-2 gene products of bovine complex I were found, suggesting that the gene cluster carrying the Paracoccus NADH dehydrogenase complex contains not only structural genes encoding water-soluble subunits but also structural genes encoding hydrophobic subunits.     

H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. Studies on topology and stoichiometry of the peripheral subunits.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane. In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P. denitrificans NDH-1 in the membranes were investigated using immunological techniques. Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes. Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex. Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic N-terminal stretch plays an important role in such an association. In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed. In addition, determination of stoichiometry of the peripheral subunits of the P. denitrificans NDH-1 was completed by radioimmunological methods. All the peripheral subunits are present as one molecule each in the enzyme complex. These results estimated the total number of cofactors in the P. denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2x[2Fe-2S] and 6x[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.     

Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans.

A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum.     

Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. When the Paracoccus NADH dehydrogenase complex was irradiated by UV light in the presence of [adenylate-32P]NAD, radioactivity was incorporated exclusively into one of three polypeptides of Mr approximately 50,000. Similar results were obtained when [adenylate-32P]NADH was used. The labeling of the Mr 50,000 polypeptide was diminished when UV irradiation of the enzyme with [adenylate-32P]NAD was performed in the presence of NADH, but not in the presence of NADP(H). The labeled polypeptide was isolated by preparative sodium dodecyl sulfate gel electrophoresis and was shown to cross-react with antiserum to the NADH-binding subunit (Mr = 51,000) of bovine NADH-ubiquinone oxidoreductase. Its amino acid composition was also very similar to that of the bovine NADH-binding subunit. These chemical and immunological results indicate that the Mr 50,000 polypeptide is an NADH-binding subunit of the Paracoccus NADH dehydrogenase complex.     

Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans

Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.     

Structural analysis of four strains of Paracoccus denitrificans

Two out of eleven newly isolated strains of Paracoccus denitrificans were investigated by light and electron microscopic methods and compared with two strains of P. denitrificans already kept in culture collections. Samples were taken from different growth phases revealing short rods and nearly spherical cells in the exponential growth phase, and an increasing ratio of nearly spherical cells in the stationary growth phase. Cell division followed the binary fission mode; higher cell aggregates were not observed. Fine structural analysis revealed extracellular surface material stainable with Ruthenium red, a gram-negative cell wall and different storage material inclusions. Structural properties and variations within the four strains under investigation are discussed and compared with those of related bacteria.     

EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase segment of the respiratory chain in Paracoccus denitrificans

The physicochemical properties of the iron-sulfur clusters present in the NADH:ubiquinone oxidoreductase of Paracoccus denitrificans have been examined in the cytoplasmic membrane particles by redox potentiometry and EPR spectroscopy. Analogous to the iron-sulfur clusters present in the mitochondrial NADH: ubiquinone oxidoreductase, we have found two binuclear and three tetranuclear EPR detectable iron-sulfur clusters, namely, N-1a, N-1b, N-2, N-3, and N-4. In the bacterial system, the two binuclear clusters differ in line shape and in Em values; the cluster with more rhombic symmetry (gx,y,z = 1.918, 1.937, 2.029) has the Em7.0 value of -150 while the almost axial one (gx,y,z = 1.929, 1.941, 2.019) has Em7.0 of -270 mV. The Em of the former cluster is pH dependent (-60 mV/pH) as in the case of mammalian N-1a while the latter is pH independent as is the mammalian cluster N-1b. The pH-dependent P. denitrificans [2Fe-2S] cluster, which we have labeled N-1a, has an Em7.0 as high as that of N-2, in contrast to the mammalian N-1a. Thus N-1a is reducible with a physiological reductant, NADH in this bacterial system. The Em of the cluster N-2 is also pH dependent (Em7.0 = -130 mV) with a pK value near 7.7. The Em values of all other clusters exhibit no pH dependence as in the case of their mammalian counterparts. We have found that the cluster N-1a is the most labile component among the five iron-sulfur clusters and may give rise to variable relative spin concentrations and extremely low Em values due to the facile modifications of the microenvironment of the cluster. The P. denitrificans NADH:ubiquinone oxidoreductase provides a unique and useful site I model system where redox composition is similar to the mitochondrial enzyme but with fewer numbers of polypeptides (Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311).     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

Exploring the membrane domain of the reduced nicotinamide adenine dinucleotide-quinone oxidoreductase of Paracoccus denitrificans: characterization of the NQO7 subunit

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.     

Paracoccus denitrificans cytochrome c1 gene replacement mutants.

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.     

Identification of the NADH-binding subunit of energy-transducing NADH-quinone oxidoreductase (NDH-1) of thermus thermophilus HB-8

The energy-transducing NADH--quinone oxidoreductase (NDH-1) isolated from Thermus thermophilus HB-8 is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN and at least three iron-sulfur clusters [Yagi, T., Hon-nami, K., and Ohnishi, T. (1988) Biochemistry 27, 2008-2013]. When NDH-1 of T. thermophilus HB-8 was irradiated by short UV light in the presence of [adenylate-32P]NADH or [adenylate-32P]NAD, radioactivity was incorporated into a single polypeptide of Mr 47,000. The labeling of the Mr 47,000 polypeptide was diminished when UV irradiation of the enzyme complex with [adenylate-32P]NAD was carried out in the presence of NADH or deamino-NADH which act as substrates for the NDH-1, but not in the presence of NADP(H) or AMP which act neither as substrates nor as competitive inhibitors. These results strongly suggest that the Mr 47,000 polypeptide is an NADH-binding subunit of the NDH-1 of T. thermophilus HB-8.     

Characterization of the 25-kilodalton subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: sequence similarity to the 24-kilodalton subunit of the flavoprotein fraction of mammalian complex I

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. Structural genes encoding the subunits of this enzyme complex constitute at least one gene cluster [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428]. The 25-kDa subunit (NQO2), which has been isolated from sodium dodecyl sulfate-polyacrylamide gels, is a polypeptide of this enzyme complex. The partial N-terminal amino acid sequence and amino acid composition of the NQO2 subunit have been determined. On the basis of the amino acid sequence, the NQO2 gene was found to be located 1.7 kilobase pairs upstream of the gene for NADH-binding subunit (NQO1). The complete nucleotide sequence of the NQO2 gene was determined. It is composed of 717 base pairs and codes for 239 amino acid residues with a calculated molecular weight of 26,122. The NQO2 subunit is homologous to the Mr 24,000 subunit of the mammalian mitochondrial NADH-ubiquinone oxidoreductase which bears an electron paramagnetic resonance-visible binuclear iron-sulfur cluster (probably cluster N1b). Comparison of the predicted amino acid sequence of the Paracoccus NQO2 subunit with those of its mammalian counterparts suggests putative binding sites for the iron-sulfur cluster. In addition, nucleotide sequencing shows the presence of two unidentified reading frames between the NQO1 and NQO2 genes. These are designated URF1 and URF2 and are composed of 261 and 642 base pairs, respectively. The possible function of the protein coded for the URF2 is discussed.     

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system.

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.