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J Biol Chem, Vol. 274, Issue 40, 28606-28611, October 1, 1999

H⁺-translocating NADH-Quinone Oxidoreductase (NDH-1) of Paracoccus denitrificans
STUDIES ON TOPOLOGY AND STOICHIOMETRY OF THE PERIPHERAL SUBUNITS^*

Takahiro Yano and Takao Yagi^§

From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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, 2×[2Fe-2S] and 6×[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Paracoccus denitrificans is a Gram-negative soil bacterium and has been called "a free-living mitochondrion" (1, 2). P. denitrificans expresses a mammalian mitochondrial type respiratory chain that bears only the proton-translocating NADH-quinone oxidoreductase (NDH-1)¹ NADH dehydrogenases (3, 4). The P. denitrificans NDH-1 is composed of at least 14 different subunits and bears one noncovalently bound FMN and at least five EPR-detectable iron-sulfur clusters as prosthetic groups (5, 6). These iron-sulfur clusters are designated cluster N1a and N1b (for [2Fe-2S] clusters) and N2, N3, and N4 (for [4Fe-4S] clusters) (6). The gene cluster encoding the P. denitrificans NDH-1 has been cloned and sequenced (7-11). The gene cluster is composed of 14 structural genes and 6 unidentified reading frames (12). These 14 structural genes have been designated nqo1 through nqo14. The NQO1 subunit contains the NADH-binding site (13), ligates cluster N3 (14), and probably bears an FMN (14). Based on the expression experiments of the putative cofactor-binding subunits, it has been suggested that the NQO2 subunit carries cluster N1a (15-17) and NQO3 subunit bears cluster N1b and N4, and probably another [4Fe-4S] cluster (18). The accompanying paper has revealed that NQO9 subunit ligates two [4Fe-4S] clusters. It is hypothesized that the NQO6 subunit may coordinate a [4Fe-4S] cluster (19). The NQO9 and NQO6 subunits are candidates for the cluster N2-binding subunit (20, 21).

Determination of subunit topology and subunit stoichiometry of the P. denitrificans NDH-1 is a prerequisite to study structure and mechanism of action of this enzyme complex. The use of subunit-specific antibodies together with membrane preparations is a reliable method for these purposes (22, 23). Previously the topological properties of the NQO4, -5, and -6 subunits of the P. denitrificans NDH-1 have been investigated (24). The NQO4, -5, and -6 subunits in membrane-bound P. denitrificans NDH-1 were extracted by treatment at alkaline pH or with chaotropes. In addition to antibodies specific to the NQO1-6 subunits (24), the antibody directed to the NQO9 subunit is available in this laboratory as shown in the accompanying paper. Therefore, it is of interest to thoroughly investigate the localization of the 7 subunits, particularly that of the NQO9 subunit, in the P. denitrificans NDH-1 in situ. In the previous study (24), the subunit stoichiometry of the NQO1-6 of the membrane-bound P. denitrificans NDH-1 has be determined to be 1 mol each per mol of the enzyme complex using radioimmunological techniques. However, there is no such information available regarding the NQO9 subunit and its homologues of other organisms. Therefore, it is of interest to elucidate the stoichiometry of the NQO9 subunit of the P. denitrificans NDH-1 because this subunit contains 2×[4Fe-4S] clusters.

In this paper we describe subunit topology and the subunit stoichiometry of the P. denitrificans NDH-1. The NQO1-6 and 9 subunits have been extracted from the P. denitrificans membranes by treatment at alkaline pH or with chaotropic agents, suggesting that these subunits are localized in the peripheral part of the P. denitrificans NDH-1 in situ. Meanwhile, we conducted the homologous expression of the NQO9 subunit of the P. denitrificans NDH-1. The N-terminal truncated and full-length NQO9 subunits were expressed, respectively, in the cytoplasm and in the cytoplasmic membrane of P. denitrificans. Based on these results the location of the NQO9 subunit as well as NQO6 subunit is discussed in this paper. In addition, subunit stoichiometry of the NQO1, -2, -4, and -9 have been determined by radioimmunoassay. The results show that there is one copy each of the NQO1-6 and -9 subunits in the P. denitrificans NDH-1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Preparation of the Sonicated and Cholate-treated P. denitrificans Membranes-- P. denitrificans membrane fraction was prepared from cells grown on glucose by lysozyme osmosis method (3). The membranes were sonicated on ice with a Brownson sonifier attached to a narrow tip at an amplitude of 5 with 50% pulse mode for 3 min. The cholate-treated P. denitrificans membranes were prepared according to Ref. 24.

Expression of the NQO1, NQO2, NQO4, and NQO9 Subunits in Escherichia coli-- The individual subunits were expressed in E. coli according to Ref. 24. pET11a(NQO1), pET11a(NQO2), pET11a(NQO4), and pET11a(NQO9), which produce full-length forms of the corresponding subunits, were used to transform E. coli strain BL21(DE3). A single colony was picked up from the plate and inoculated into 30 ml of 2× YT medium containing 100 µg/ml ampicillin and was grown at 37 °C. When A₆₀₀ reached 0.5, isopropyl--D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the cells were cultivated at 37 °C for 4 h. The cells were harvested in a GSA rotor at 6,000 rpm for 10 min.

Preparation of Inclusion Body Fractions-- Inclusion body fractions were prepared according to Ref. 24. The cells were suspended in 10 mM Tris-HCl buffer (pH 8.0) containing 0.3 M NaCl, 1.0 mM DTT, 1.0 mM EDTA, and 0.1 mM PMSF. The cell suspension was freeze-thawed twice with liquid nitrogen and twice with a 30 °C water bath. The resulting cell suspension was sonicated on ice with a Brownson sonifier attached to a narrow tip at an amplitude of 7 with 50% pulse mode for 10 min three times. The suspension was centrifuged, and the inclusion bodies were recovered. The inclusion bodies were washed with 50 mM Tris-HCl buffer (pH 8.0) containing 10%(w/v) sucrose, 1.0 mM DTT, 1.0 mM EDTA, and 0.1 mM PMSF by repeating homogenization and centrifugation three times. The inclusion bodies were further washed with 50 mM Tris-HCl buffer (pH 8.0) containing 2% (w/v) Triton X-100, 1.0 mM DTT, 1.0 mM EDTA, and 0.1 mM PMSF in the same way. After these treatments, purity of the inclusion bodies was assessed to be more than 90% on the basis of SDS-PAGE analysis.

Purification of the Inclusion Bodies by Chromatography-- The inclusion bodies obtained were solubilized in 6 M urea buffer containing 10 mM Tris-HCl (pH 8.0), 1.0 mM DTT, 1.0 mM EDTA, and 0.1 mM PMSF and stirred at 4 °C overnight. The protein solutions were cleared by centrifugation in an SS34 rotor at 12,000 rpm for 15 min, and the supernatants were recovered. The solubilized subunits were applied onto a DEAE-Toyopearl column (3.0 × 10 cm) equilibrated with the same buffer. The column was extensively washed with the equilibration buffer, and the absorbed proteins were eluted with a linear gradient of NaCl (0-0.3 M) in the same buffer. Typically, the subunits were eluted as a single peak. The fractions were pooled, and the purity was checked by SDS-PAGE. Usually the subunits thus obtained were electrophoretically homogeneous. If further purification was needed, the following chromatography was carried out. The protein fractions were dialyzed against 1 liter of 6 M urea buffer containing 10 mM Tris-HCl (pH 8.0), 1.0 mM DTT, 1.0 mM EDTA, and 0.1 mM PMSF overnight. The solutions were applied onto a DEAE-Bio-Gel column (1.5 × 10 cm) equilibrated with the same buffer. The column was washed with the equilibration buffer, and the absorbed proteins were eluted by a linear gradient of NaCl (0-0.3 M) in the same buffer. The fractions were collected and used for the experiments.

Construction of the Plasmid of the Truncated Form of the NQO9 Subunit for Expression in P. denitrificans-- For the expression in P. denitrificans a broad range host plasmid pEG400 was used (25). In order to express the nqo9 gene, we used a promoter region of cycA gene that encodes cytochrome c₅₅₀ (26). The promoter is known to be active in a regulative manner depending upon energy sources on which the cells grow (27). The promoter region (~240 base pairs) was amplified by polymerase chain reaction method with the following oligonucleotides: cycAF, 5'-GGA TCC TCT AGA GTC GAC ATG GGC CTG CC-3'; cycAR, 5'-CAT ACA TAT GAT CTT CAT CGC GTT TCC TC-3'. The forward primer, cycAF, was designed to have BamHI (GGATCC) and XbaI (TCTAGA) in addition to SalI (GTCGAC) at the 5' end for the convenience of DNA manipulation (underlined letters). Italic letters indicated altered sequences from the original. The reverse primer, cycAR, contains an NdeI site (CATATG, underlined letters) 6 base pairs downstream from the translation initiation codon keeping the first methionine and alanine in frame. Altered sequences were indicated in italic. In other words, a gene of interest is expressed under the cycA promoter having extra amino acid sequences, Met-Ala, at its N-terminal end. The polymerase chain reaction-amplified DNA segment was subcloned in pCR-script II (Stratagene), and its sequences were verified by sequencing of both strands. A clone containing the cycA promoter region in a particular direction was chosen and designated as pCR(PcycA). The pCR(PcycA) was digested with SalI and BamHI and re-cloned in SalI and BamHI sites of pTZ18U and named pTZ18(PcycA). Meanwhile, one of the NQO9 plasmids such as pCR(NQO91-30) was digested with NdeI and BamHI and the DNA fragments containing the nqo9 gene were then ligated in the NdeI and BamHI sites of the pTZ18(PcycA) prepared above. Clones were selected and designated pTZ18(PcycA-NQO91-30). The pTZ18(PcycA-NQO9 1-30) was subsequently digested with EcoRI and PstI, and the DNA fragments containing the cycA promoter + nqo9 gene were ligated at EcoRI and PstI sites of pEG400. In this way, three plasmids were constructed and designated pEG400(PcycA-NQO9 1-30), pEG400(PcycA-NQO9 1-30, 151-163), and pEG400(PcycA-NQO9 1-30, 141-163) and used for expression study.

Expression of the Truncated Form of the NQO9 Subunit in P. denitrificans and Preparation of the Cytoplasmic Fraction-- The expression plasmids described above were transferred into P. denitrificans strain Pd1222 by conjugation through E. coli strain SM10 according to Ref. 26. The transformed P. denitrificans cells were selected on a brain heart infusion (BHI) plate containing 40 µg/ml rifampicin and 25 µg/ml streptomycin. A single and well isolated colony was picked up and spread onto a new plate containing the same antibiotics. The transformants were grown in liquid BHI medium to late exponential phase. The cell pellets were suspended in 10 mM Tris-HCl buffer (pH 8.0) containing 1.0 mM DTT, 1.0 mM EDTA, and 0.1 mM PMSF to be approximately 20% (w/v). The cell suspensions were freeze-thawed twice with liquid nitrogen and a 30 °C water bath and sonicated with a Brownson sonifier attached to a narrow tip at an amplitude of 5 with 50% pulse mode for 5 min. The suspensions were centrifuged at 10,000 rpm for 10 min in an SS34 rotor to separate from unbroken cells. The resultant cell-free extracts were then ultracentrifuged at 50,000 rpm for 60 min in 60Ti rotor. The supernatants were carefully recovered and subjected to Western analysis.

Quantitative Immunoblotting-- Quantitative immunoblotting was carried out according to Hekman et al. (23). The amounts of bound primary antibodies were detected with ¹²⁵I-protein A (250,000 cpm/ml) dissolved in 1× phosphate-buffered saline containing 2% (w/v) skim milk after incubation with the PVDF membranes for 1 h at room temperature. The membranes were washed with 1× phosphate-buffered saline containing 0.3% (w/v) Tween 20 for 10 min three times. The membranes were air-dried and exposed to Fuji medical x-ray film overnight at room temperature. Radioactive bands were then excised from the membranes, and the radioactivity associated with each band was determined by a -counter.

Other Analytical Procedures-- Protein was estimated by the method of Lowry et al. (28) in the presence of 1 mg/ml potassium deoxycholate (29). SDS-polyacrylamide gel electrophoresis was carried out by the method of Laemmli (30). Amino acid composition analysis (13), electroblotting onto PVDF membranes (31), and immunoblotting (32-34) were performed according to the references cited. Any variations from the procedures and other details are described in the figure legends.

Materials-- Acrylamide, N,N'-methylenebis(acrylamide), SDS, SDS-PAGE marker proteins, Coomassie Brilliant Blue R-250, DEAE-Bio-Gel were from Bio-Rad. Horseradish peroxidase-conjugating affinity purified antibodies to rabbit IgG and ECL kit were from Amersham Pharmacia Biotech. DEAE-Toyopearl resin was from Tosohaas. Expression vector, pET11a, and E. coli strain BL21(DE3) were from Novagen. A broad host-range plasmid, pEG400, was kindly supplied by Dr. Bernd Ludwig (Johann Wolfgang Goethe-Universität, Germany). A clone containing cycA gene, P. denitrificans strain, Pd1222, and E. coli strain SM10 were generous gifts from Dr. Rob van Spanning (Vrije Universiteit, The Netherlands).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Localization of the Peripheral Subunits in the P. denitrificans NDH-1-- In the present study, we thoroughly investigated topological properties of 7 subunits (NQO1, NQO2, NQO3, NQO4, NQO5, NQO6, and NQO9) of the P. denitrificans NDH-1. Although function of the individual subunits is not yet fully understood, it seems likely that the electron transfer reaction from NADH takes place through these subunits since the redox components, FMN, and all iron-sulfur clusters reside in them. Of particular interest is the localization of the NQO6 and NQO9 subunits in NDH-1. One of these subunits is postulated to bear the cluster N2 that plays a key role in electron transfer to quinone. We conducted extraction experiments of the subunits with several chaotropic reagents or alkaline solutions and subunit-specific antibodies. Treatment with these reagents has been known to disrupt protein-protein interactions to some extent and to extract extrinsic proteins from membranes (35, 36). Although the structure of the enzyme complex at an atomic level is not yet available, information obtained in these experiments is still beneficial to understanding the rough structure of the enzyme complex. Some of the results have been reported previously concerning the NQO4, NQO5, and NQO6 subunits (24). When the membrane-embedded NDH-1 enzyme complex was treated with NaI, all the subunits were extracted into the supernatants (Fig. 1), whereas treatment with a high concentration of NaCl scarcely extracted the subunits. A noticeable observation is that the extent of extraction with NaBr varied among the subunits. The NQO1 and NQO2 were largely extracted, whereas the NQO3 and NQO4 were moderately extracted, and the release of the NQO5, NQO6, and NQO9 subunits from the membranes was slight. Moreover, when urea was used, the NQO6 and NQO9 remained in the membranes, whereas the other subunits were almost completely extracted. When the membranes were incubated in alkaline buffer, all subunits could be extracted from membranes to greater extents as pH increased from 10 to 12 (Fig. 1). These results demonstrate that all the subunits are extractable from the membranes, but the extraction of the NQO6 and NQO9 subunits is less effective than the other subunits. It can be speculated, therefore, that the NQO6 and NQO9 subunits are directly associated with the membrane part of the complex (NQO7, NQO8, and NQO10-14 subunits). Other groups have also expressed a similar view (37, 38). Of interest is that the topological character of the NQO9 subunit found in this study seems to be compatible with the results of expression studies reported in the accompanying paper and also see Ref. 18. The full-length form of NQO9 was expressed in the cytoplasmic membranes in E. coli, whereas the deletion of the hydrophobic N-terminal stretches made the subunit completely water-soluble. We attempted to express these truncated forms of the NQO9 subunit in P. denitrificans itself in order to see whether the same phenomena could be observed. We utilized a promoter region of the cycA gene that encoded cytochrome c₅₅₀. The nqo9 genes encoding truncated subunits NQO9(1-30), NQO9(1-30, 151-163), and NQO9(1-40, 151-163) were linked under a cycA promoter on a broad host-range plasmid, pEG400, respectively. The constructs were transferred into P. denitrificans strain Pd1222 through E. coli strain SM10 by conjugation (see "Experimental Procedures"). Transformants were isolated on BHI plates containing streptomycin + rifampicin and were aerobically grown in BHI liquid medium. In order to avoid undesired recombination events, experiments were conducted with freshly transformed cells each time. Cytoplasmic fractions were prepared from individual transformants and subjected to Western blotting analysis. The truncated subunits could be readily distinguished from the native form of the NQO9 subunit by the difference in molecular size. It is clear in Fig. 2 that all truncated forms of the NQO9 subunit were expressed in cytoplasm of P. denitrificans in a manner similar to the expression in E. coli (see the accompanying paper). It is noteworthy that the hydrophobic N-terminal region of the NQO9 subunit and its homologues (corresponding to 30 amino acid residue long of the P. denitrificans NQO9 subunit) is predicted to form -helix (data not shown). It can be speculated, therefore, that the hydrophobic N-terminal stretches of the NQO9 subunit may play an important role in its association with the membrane part of the enzyme complex in a manner similar to the Rieske iron-sulfur subunit of ubiquinol-cytochrome c oxidoreductase complex. Although the Rieske iron-sulfur subunit is extractable from membranes by the same treatment used in this study, its hydrophobic and less conserved N-terminal region has been found to lie in the membrane together with -helical bundles of the cytochrome b subunit as depicted by x-ray structure (39, 40). The proteolytic cleavage or genetic deletion of the N-terminal stretch of the Rieske iron-sulfur cluster subunit of mitochondrial and bacterial ubiquinol-cytochrome c oxidoreductase complex have resulted in the recovery or expression of soluble forms of the iron-sulfur cluster domain (41). Further experiments are needed to examine this notion in the future. On the other hand, information concerning the NQO6 subunit is limited. The NQO6 subunit is also relatively hydrophobic. The expressed NQO6 subunit is localized in the membrane of E. coli in a manner similar to the case of the NQO9 subunit (24), and the products can be extracted from the membranes only in the presence of detergents.² Further characterization of the subunit is underway in our laboratory.

View larger version (82K): [in this window] [in a new window]   Fig. 1.   Effect of chaotropic reagents and alkaline buffer on extraction of the NQO1, NQO2, NQO3, NQO4, NQO5, NQO6, and NQO9 subunits from the cholate-treated P. denitrificans membranes. Salt concentrations used were 6 M urea, 2 M NaI, 2 M NaBr, and 2 M NaCl. The P. denitrificans membranes were suspended in 100 mM CHES buffer (pH 10, 11, and 12). S, the supernatant fractions; M, the membrane fractions. The detailed extraction procedures were described under "Experimental Procedures." Immunoblotting was carried out using antibodies against the expressed NQO1, NQO2, NQO3, NQO4, NQO5, NQO6, and NQO9 subunits and horseradish peroxidase-conjugated anti-rabbit IgG antibody as described (32-34) except that the detection was performed with an ECL kit (Amersham Pharmacia Biotech).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Expression of the truncated forms of the NQO9 subunit in P. denitrificans. Approximately 3 µg of the cytoplasmic proteins prepared from P. denitrificans cells harboring the expression plasmids were loaded on 12% Schägger's SDS-polyacrylamide gel and analyzed by Western blotting in the same way as Fig. 1. Lane 1, NQO9(1-30); lane 2, NQO9(1-30,151-163); lane 3, NQO9(1-30,141-163). A small quantity of the NQO9 subunit contaminated from the membrane fractions was detected (marked by asterisk).

Stoichiometry of the Peripheral Subunits of the P. denitrificans NDH-1-- It is important to determine the stoichiometric ratios of all the subunits constituting the NDH-1 enzyme complex since such information will provide the structural basis to clarify the mechanism of action of the enzyme complex. Particularly, determination of the copy number of the cofactor-binding subunits in the enzyme complex will make it possible to estimate the number of cofactors at a protein level. Many attempts have been made in the past to determine the number of cofactors in the mitochondrial complex I by analyzing the contents of FMN, non-heme iron, and acid-labile sulfide (42, 43). However, a clear answer has not yet been found mainly due to technical difficulties and some intrinsic problems associated with the materials used. In the previous study, the stoichiometric ratios of the NQO1-6 subunits of P. denitrificans NDH-1 have been determined by a radioimmunological method (24). This method is known to be reliable for estimating the quantity of particular polypeptides in crude preparations or multi-subunit enzyme complexes (23, 44). We employed the same approach to determine the stoichiometry of the NQO9 subunit in this study. Contents of the NQO9 subunit as well as NQO1, NQO2, and NQO4 subunits in the cholate-treated P. denitrificans cytoplasmic membranes were determined. The individual subunits were expressed in E. coli, and the polypeptides were homogeneously purified and used as standard proteins (Fig. 3). In order to determine the correct stoichiometric ratio of the subunits in the enzyme complex, a painstaking effort was made to determine the protein concentrations as accurately as possible. Two methods were employed. The protein concentrations were determined for each standard subunit solution by Lowry's method and corrected using values obtained by amino acid composition analysis. Known amounts of the individual standard subunits and P. denitrificans membranes were loaded on a Laemmli's SDS-polyacrylamide gel, and the proteins were transferred onto PVDF membranes. The immobilized proteins were quantitated on the membranes by radioimmunoassay as described under "Experimental Procedures."

View larger version (29K): [in this window] [in a new window]   Fig. 3.   SDS-polyacrylamide gel pattern of the expressed and purified NQO1, NQO2, NQO4, and NQO9 subunits of the P. denitrificans NDH-1. The subunits were loaded onto a 13% Laemmli's gel. The gel was stained with Coomassie Brilliant Blue and destained in 7% acetic acid solution. M, molecular size marker (97, 66, 45, 31, 21.5, and 14.5 kDa); lane 1, NQO1 subunit; lane 2, NQO2 subunit; lane 4, NQO4 subunit; and lane 9, NQO9 subunit.

Fig. 4 depicts one of the examples where the NQO9 subunits were detected by autoradiography after incubation with anti-NQO9 subunit antiserum followed by ¹²⁵I-protein A. Quantitation of bound ¹²⁵I relative to the amounts of loaded proteins gave reasonable linear relationships in ranges of 0-20 ng for the standard proteins and 0-1.0 µg for the P. denitrificans membranes. By comparing the slopes of the lines between the standard and experimental plots, the contents of the individual subunits in the membranes (nanomoles of subunit/mg of protein) were calculated. The stoichiometric ratios of the subunits relative to NQO1 were then obtained (Table I). The stoichiometric ratio of NQO9 subunit to NQO1 subunit was found to be 0.95, whereas those of the NQO2 and NQO4 subunits were 0.97 and 1.04, respectively. The results of the NQO2 and NQO4 subunits are in a good agreement with the previous report (24). Since it is believed that the mitochondrial complex I and bacterial NDH-1 are present as a monomer (20, 45, 46), it seems likely that these 7 peripheral subunits are all present as one molecule each in the enzyme complex (Table I). These results also allow us to estimate the total number of the cofactors, considering the fact that FMN and all iron-sulfur clusters are located in these subunits. It seem likely that the P. denitrificans NDH-1 enzyme complex contains one molecule of FMN and up to 8 iron-sulfur clusters, 2×[2Fe-2S] and 6×[4Fe-4S] (Table II). Mitochondrial complex I and Rhodobacter capsulatus NDH-1 may contain the same number of cofactors as P. denitrificans NDH-1 because they are predicted to have the same number of cofactor-binding sites (21, 46). In the case of E. coli and Thermus thermophilus NDH-1, the total number of iron-sulfur clusters can be 9 since they seem to contain an additional [2Fe-2S] cluster that is tentatively designated cluster N1c (also see Table II) (47, 48).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   A, autoradiograms of immunoblotting of the purified NQO9 subunit of the P. denitrificans NDH-1 (left) and the cholate-treated P. denitrificans membranes (Pd, right) using NQO9 subunit-specific antiserum. B, standard curve (left) and experimental curve (right) for quantitative immunoblotting for the NQO9 subunit. Purified NQO9 subunit and the cholate-treated P. denitrificans membranes were loaded onto a 13% SDS-polyacrylamide gel and transferred onto PVDF membranes according to Matsudaira (31). The membranes were incubated with anti-NQO9 antiserum followed by 125I-protein A as described under "Experimental Procedures." After radioactive bands were located, they were excised from the membranes and counted in a -counter.

Conclusion-- In this study, topology and stoichiometry of the 7 peripheral subunits of the P. denitrificans NDH-1 were investigated. It seems likely that the P. denitrificans NDH-1 enzyme complex contains one FMN and up to 8 iron-sulfur clusters. The extraction experiments have suggested that the NQO6 and NQO9 subunits are directly associated with the membrane part of the enzyme complex, constituting a junction between the peripheral and membrane portions as shown in Fig. 5. In this connection, it has been suggested that the hydrophobic N-terminal stretch of the NQO9 subunit plays a structurally important role. Recently, the NQO6 subunit and its mitochondrial counterpart, PSST subunit, have been identified as a conserved specific binding site for very hydrophobic complex I inhibitors such as pyridaben, rotenone, and piericidin A (49). Our results described here are also consistent with those findings with respect to the fact that the subunit is partially surrounded by a hydrophobic environment. Both NQO6 and NQO9 subunits appear to be important for the energy transduction at site 1. It is conceivable, therefore, that further characterization of these subunits will be a crucial step toward the elucidation of the mechanism of action of the enzyme complex.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Schematic presentation of the structural model of the P. denitrificans NDH-1 enzyme complex in the cytoplasmic membrane. Exact configuration of the subunits and copy number of the membrane subunits are not yet known.

	ACKNOWLEDGEMENTS

We thank Julieann Grant and Karina Lichtenstein for their excellent technical assistance and Drs. Salvatore Di Bernardo, Byoung Boo Seo, and Akemi Matsuno-Yagi for discussion. We are grateful to Prof. Tomoko Ohnishi and Dr. Carla Hekman for critical reading of the manuscript. Computer facilities were supported by United States Public Health Service Grant M01RR00833 for the General Clinical Research Center. Synthesis of oligonucleotides was supported in part by the Sam and Rose Stein Endowment Fund.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant R01GM33712. This is publication 12275-MEM from The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104.

^§ To whom correspondence should be addressed. E-mail: yagi@ scripps.edu.

² T. Yano, T. Ohnishi, and T. Yagi, unpublished results.

	ABBREVIATIONS

The abbreviations used are: NDH-1, bacterial proton-translocating NADH-quinone oxidoreductases; complex I, mitochondrial proton-translocating NADH-quinone oxidoreductase; DTT, dithiothreitol; FMN, flavin mononucleotide; PMSF, phenylmethanesulfonyl fluoride; PVDF, polyvinylidene difluoride; BHI, brain heart infusion; CHES, 2-(cyclohexylamino)ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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