NADH dehydrogenases: from basic science to biomedicine.

This review article is concerned with two on-going research projects in our laboratory, both of which are related to the study of the NADH dehydrogenase enzyme complexes in the respiratory chain. The goal of the first project is to decipher the structure and mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from two bacteria, Paracoccus denitrificans and Thermus thermophilus HB-8. These microorganisms are of particular interest because of the close resemblance of the former (P. denitrificans) to a mammalian mitochondria, and because of the thermostability of the enzymes of the latter (T. thermophilus). The NDH-1 enzyme complex of these and other bacteria is composed of 13 to 14 unlike subunits and has a relatively simple structure relative to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which is composed of at least 42 different subunits. Therefore, the bacterial NDH-I is believed to be a useful model for studying the mitochondrial complex I, which is understood to have the most intricate structure of all the membrane-associated enzyme complexes. Recently, the study of the NADH dehydrogenase complex has taken on new urgency as a result of reports that complex I defects are involved in many human mitochondrial diseases. Thus the goal of the second project is to develop possible gene therapies for mitochondrial diseases caused by complex I defects. This project involves attempting to repair complex I defects in the mammalian system using Saccharomyces cerevisiae NDI1 genes, which code for the internal, rotenone-insensitive NADH-quinone oxidoreductase. In this review, we will discuss our progress and the data generated by these two projects to date. In addition, background information and the significance of various approaches employed to pursue these research objectives will be described.

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III.

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing

Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells.

The Ndi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells (Seo, B. B., Kitajima-Ihara, T., Chan, E. K., Scheffler, I. E., Matsuno-Yagi, A., and Yagi, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9167-9171) and human embryonal kidney 293 (HEK 293) cells (Seo, B. B., Matsuno-Yagi, A., and Yagi, T. (1999) Biochim. Biochem. Acta 1412, 56-65) and that the Ndi1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I). To extend the potential use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1). With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1. The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells. Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1-transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated. The Ndi1 protein has a great potential as a molecular remedy for complex I defects, and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.

Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae.

In contrast to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed.

The NDUFA1 gene product (MWFE protein) is essential for activity of complex I in mammalian mitochondria.

The MWFE polypeptide of mammalian complex I (the proton-translocating NADH-quinone oxidoreductase) is 70 amino acids long, and it is predicted to be a membrane protein. The NDUFA1 gene encoding the MWFE polypeptide is located on the X chromosome. This polypeptide is 1 of approximately 28 "accessory proteins" identified in complex I, which is composed of 42 unlike subunits. It was considered accessory, because it is not one of the 14 polypeptides making up the core complex I; a homologous set of 14 polypeptides can make a fully functional proton-translocating NADH-quinone oxidoreductase in prokaryotes. One MWFE mutant has been identified and isolated from a collection of respiration-deficient Chinese hamster cell mutants. The CCL16-B2 mutant has suffered a deletion that would produce a truncated and abnormal MWFE protein. In these mutant cells, complex I activity is reduced severely (<10%). Complementation with hamster NDUFA1 cDNA restored the rotenone-sensitive complex I activity of these mutant cells to approximately 100% of the parent cell activity. Thus, it is established that the MWFE polypeptide is absolutely essential for an active complex I in mammals.

Ubiquinol:cytochrome c oxidoreductase. Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b.

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N', N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0. 06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.

Molecular remedy of complex I defects: rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells.

The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable NDI1-transfected cells were obtained by screening with antibiotic G418. The NDI1 gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunofluorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate. The activities were inhibited by flavone, antimycin A, and KCN but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes. Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the NDI1-transfected cells were able to grow in the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1. These results indicated that the S. cerevisiae Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

Ubiquinol:cytochrome c oxidoreductase. The redox reactions of the bis-heme cytochrome b in unenergized and energized submitochondrial particles.

The redox reactions of the bis-heme cytochrome b of the ubiquinol:cytochrome c oxidoreductase complex (complex III, bc1 complex) were studied in bovine heart submitochondrial particles (SMP). It was shown that (i) when SMP were treated with the complex III inhibitor myxothiazol (or MOA-stilbene or stigmatellin) or with KCN and ascorbate to reduce the high potential centers of complex III (iron-sulfur protein and cytochromes c + c1), NADH or succinate reduced heme bL slowly and incompletely. In contrast, heme bH was reduced by these substrates completely and much more rapidly. Only when the complex III inhibitor was antimycin, and the high potential centers were in the oxidized state, NADH or succinate was able to reduce both bH and bL rapidly and completely. (ii) When NADH or succinate was added to SMP inhibited at complex III by antimycin and energized by ATP, the bis-heme cytochrome b was reduced only partially. Prereduction of the high potential centers was not necessary for this partial b reduction, but slowed down the reduction rate. Deenergization of SMP by uncoupling (or addition of oligomycin to inhibit ATP hydrolysis) resulted in further b reduction. Addition of ATP after b was reduced by substrate resulted in partial b oxidation, and the heme remaining reduced appeared to be mainly bL. Other experiments suggested that the redox changes of cytochrome b effected by energization and deenergization of SMP occurred via electronic communication with the ubiquinone pool. These results have been discussed in relation to current concepts regarding the mechanism of electron transfer by complex III.

Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis.

Mitochondrial function was examined in Jurkat cells undergoing Fas-mediated apoptosis. With succinate or ascorbate/tetramethylphenylenediamine as substrate, oxygen uptake by digitonin-permeabilized apoptotic mitochondria was greatly decreased as compared with control. Assessment of the function of the cytochrome c-cytochrome oxidase segment of the electron transport chain of apoptotic mitochondria showed that the activity of cytochrome oxidase appeared to be normal, but that of cytochrome c was greatly diminished. A death protease was found to participate in the events leading to the loss of cytochrome c activity, but the cytochrome did not seem to be extensively degraded during the course of apoptosis. Our results suggest that a rapid loss in mitochondrial function due at least in part to the inhibition or inactivation of cytochrome c is a potentially fatal component of the apoptosis program of Jurkat cells.

Ubiquinol-cytochrome c oxidoreductase. The redox reactions of the bis-heme cytochrome b in ubiquinone-sufficient and ubiquinone-deficient systems.

Antimycin and myxothiazol are stoichiometric inhibitors of complex III (ubiquinol-cytochrome c oxidoreductase), exerting their highest degree of inhibition at I mol each/mol of complex III monomer. Phenomenologically, however, they each inhibit three steps in the redox reaction of the bis-heme cytochrome b in submitochondrial particles (SMP), and all three inhibitions are incomplete to various extents. (i) In SMP, reduction of hemes bH and bL by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol. Each inhibitor alone allows reduced bH and bL to accumulate, indicating that each inhibits the reoxidation of these hemes. (E)-Methyl-3-methoxy-2-(4')-trans-stilbenyl)acrylatc in combination with antimycin or 2-n-heptyl-4-hydroxyquinoline-N-oxide in combination with myxothiazol causes less inhibition of b reduction than the combination of antimycin and myxothiazol. (ii) Reoxidation of reduced b, is inhibited by either antimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline-N-oxide, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate, or stigmatellin). (iii) Reoxidation of reduced bH is also inhibited by any one of these reagents. These inhibitions are also incomplete, and reduced bL is oxidized through the leaks allowed by these inhibitors at least 10 times faster than reduced bH. Heme bH can be reduced in SMP via cytochrome c, and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). Energization of SMP by the addition of ATP affords reduction of bL as well. Reverse electron transfer to bH and bL is inhibited partially by myxothiazol, much more by antimycin. Ascorbate + TMPD also reduce bH in ubiquinone-extracted SMP in which the molar ratio of ubiquinone to cytochrome b has been reduced 200-fold from 12.5 to aproximately 0.06. Reconstitution of the extracted particles with ubiquinone-10 restores substrate oxidation but does not improve the rate or the extent of b, reduction by ascorbate + TMPD. These reagents also partially reduce cytochrome b in SMP from a ubiquinone-deficient yeast mutant. The above results are discussed in relation to the Q-cycle hypothesis.

Characteristics of the energy-transducing NADH-quinone oxidoreductase of Paracoccus 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) of Paracoccus 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 the Paracoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding the Paracoccus 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 and Paracoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.

Studies on the mechanism of oxidative phosphorylation. ATP synthesis by submitochondrial particles inhibited at F0 by venturicidin and organotin compounds.

Oligomycin,N,N'-dicyclohexylcarbodiimide (DCCD), venturicidin, and tetracoordinate organotin compounds (R3SnX) are potent inhibitors of the mitochondrial ATP synthase complex, all acting on the membrane sector, F0. Oligomycin and DCCD inhibit proton translocation through F0 and energy transfer between F0 and the catalytic sector, F1, of the ATP synthase complex. Our results have shown that venturicidin and organotin compounds (tributyltin and triphenyltin chloride were used) greatly attenuate these processes, but do not cause complete inhibition. As a result, bovine submitochondrial particles (SMP) treated with venturicidin or tributyltin chloride were shown to be capable of ATP hydrolysis and synthesis, albeit at very slow rates. We had shown previously that in ATP synthesis Vmax and apparent Km for ADP and Pi increase or decrease, respectively, as the steady-state membrane potential is elevated or lowered (Matsuno-Yagi, A., and Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038). These changes occurred at constant Vmax/Km, suggesting that the apparent Km changes were due mainly to kcat changes. Results presented here show that, in respiring SMP treated with venturicidin or organotin compounds, the membrane potential is near the static-head level, but the slow rate of ATP synthesis takes place with a low KmADP value of 2-3 microM. In agreement with our previous conclusions, these results indicate that it is not the membrane potential per se that affects KmADP during ATP synthesis, but rather it is the rate of energy transfer from F0 to F1 that influences both Vmax and KmADP. Further conclusions from the above studies have been discussed in relation to the possible mechanism of energy transfer between F0 and F1 and the manner in which venturicidin and organotin compounds might attenuate this process.

DNA sequencing of the seven remaining structural genes of the gene cluster encoding the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans.

In our previous papers, seven structural genes (NQO1-7) of the energy-transducing NADH-quinone (Q) oxidoreductase of Paracoccus denitrificans were characterized [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991a) Biochemistry 30, 8678-8684; (1991b) Biochemistry 30, 6422-6428; (1992a) Biochemistry 31, 6925-6932; (1992b) Arch. Biochem. Biophys. 296, 40-48]. This paper reports the identification, cloning, and sequencing of seven additional structural genes in the same gene cluster (P. denitrificans enzyme complex). These seven genes, designated NQO8-14, are composed of 1038, 492, 603, 306, 2112, 1542, and 1500 base pairs, respectively. The polypeptides encoded by the NQO8-14 genes are homologous, respectively, to the ND1 product, the 23-kDa polypeptide, and the ND6, ND4L, ND5, ND4, and ND2 products of the bovine NADH-Q oxidoreductase. The order of the 14 structural genes of the Paracoccus energy-transducing NADH-Q oxidoreductase in the gene cluster is NQ07, NQO6, NQO5, NQO2, NQO1, NQO3, NQO8, NQO9, NQO10, NQO11, NQO12, NQO13, and NQO14. Downstream from the NQO14 gene an open reading frame (designated URF240) was detected which encodes a predicted polypeptide homologous to the biotin [acetyl-CoA-carboxylase] ligase of Escherichia coli. In addition, a putative terminal sequence motif was observed downstream of the NQO14 gene, suggesting that the structural gene NQO14 is the 3'-terminal gene of the Paracoccus NADH-Q oxidoreductase gene cluster. Nucleotide sequencing of the entire gene cluster revealed the presence of three unidentified reading frames: one between the NQO3 and NQO8 genes and other two between the NQO9 and NQO10 genes. These are designated URF4, URF5, and URF6 and are composed of 768, 393, and 405 base pairs, respectively. The possible functions of the putative proteins encoded by URF5 and URF6 are discussed.

Studies on the mechanism of oxidative phosphorylation. Different effects of F0 inhibitors on unisite and multisite ATP hydrolysis by bovine submitochondrial particles.

Bovine submitochondrial particles prepared in the presence of GTP (G-SMP), as well as G-SMP washed in 150 mM KCl, catalyzed unisite ATP hydrolysis with a first order rate constant of 0.12 s-1. This rate constant remained unchanged at ATP concentrations < 0.06 microM but increased sharply at higher ATP concentrations, presumably because of ATP binding to other catalytic or regulatory sites. Pretreatment of the particles with oligomycin greatly inhibited unisite ATP binding, in agreement with previous findings. Pretreatment of the particles with N,N'-dicyclohexylcarbodiimide had a slight effect on unisite ATP binding, whereas pretreatment with the inhibitors venturicidin and tributyl(or triphenyl)tin chloride had no effect. Titration of unisite ATPase activity with increasing concentrations of oligomycin or efrapeptin resulted in sigmoidal inhibition curves, as though more than a single inhibition site was being titrated by each inhibitor. Venturicidin and organotin compounds had little effect on the ATPase activity of SMP at [ATP] < or = [F1] and did not cause 100% inhibition at [ATP] >> [F1]. By analogy to our previous studies on the inhibition of the ubiquinol-cytochrome c reductase complex by antimycin (Hatefi, Y., and Yagi, T. (1982) Biochemistry 24, 6614-6618), it is proposed that venturicidin and organotin compounds freeze the structure of the F0 sector of the ATP synthase complex in such a manner that prevents the subunit molecular motions required for rapid proton flux but allows a slow proton flux generated by ATPase activity at low ATP concentrations.

Gene cluster of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans: characterization of four structural gene products.

In previous reports from our laboratory, the three structural genes (NQO1, NQO2, and NQO3) of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans were characterized [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428; (1991) Biochemistry 30, 8678-8684; (1992) Arch. Biochem. Biophys. 296, 40-48]. In this report, the four structural genes NQO4, NQO5, NQO6, and NQO7 of the same Paracoccus denitrificans oxidoreductase were cloned and sequenced. On the basis of sequence homology and immunological cross-reactivity, these genes encode counterparts of the 49-, 30-, and 20-kDa polypeptides and the mitochondrial DNA ND3 polypeptides of bovine mitochondrial complex I. These seven structural genes were found to be located in the same gene cluster. The order of the seven structural genes of the Paracoccus NADH-quinone oxidoreductase in the gene cluster is NQO7, NQO6, NQO5, NQO4, NQO2, NQO1, and NQO3. Upstream of the NQO7 gene, an open reading frame encoding a predicted polypeptide homologous to the UV repair enzyme A of Escherichia coli and Micrococcus lysodeikticus was detected. The 5'-terminus of the gene cluster carrying the Paracoccus NADH-quinone oxidoreductase was studied, and the possible promoter region is discussed. The NQO4 and NQO5 genes appear to code for the M(r) 48,000 and 21,000 polypeptides of the isolated Paracoccus NADH dehydrogenase complex [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311] on the basis of amino acid analyses and N-terminal protein sequence analyses. The antisera to the bovine complex I 49- and 30-kDa polypeptides cross-reacted with the Paracoccus 48- and 21-kDa subunits, respectively.