Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Characterization of the membrane domain subunit NuoJ (ND6) of the NADH-quinone oxidoreductase from Escherichia coli by chromosomal DNA manipulation

The ND6 subunit is one of seven mitochondrial DNA-encoded subunits of the proton-translocating NADH-quinone oxidoreductase (complex I). Physiological importance of the ND6 subunit is becoming increasingly apparent because a number of mutations leading to amino acid changes in this subunit have been found to be associated with known mitochondrial diseases. Using the Escherichia coli enzyme (NDH-1), we have investigated the NuoJ subunit (the E. coli counterpart of ND6) by employing a chromosomal DNA manipulation technique. A series of point mutations was constructed directly on the nuoJ gene in the chromosome targeting at highly conserved residues. Analyses with blue-native gel electrophoresis and immunological methods revealed that, in all point mutants, the assembly of NDH-1 was normal and that the deamino-NADH-K(3)Fe(CN)(6) reductase activity of the membrane was essentially the same as that of the wild-type. However, energy-coupled NDH-1 activities were affected to varied extents. Among them, mutants of the Val-65 residue that is located in the most conserved transmembrane segment significantly lost the coupled electron-transfer activities and exhibited diminished membrane potential and proton translocation. This may suggest that Val-65 or the area around it is important for energy transduction of the coupling site 1. Together with the results on mutations related to human diseases, possible functional roles of the NuoJ subunit have been discussed.     

Pivotal roles of three conserved carboxyl residues of the NuoC (30k) segment in the structural integrity of proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The prokaryotic proton-translocating NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzyme that contains 14 subunits (NuoA-NuoN or Nqo1-Nqo14). All subunits have their counterparts in the eukaryotic enzyme (complex I). NDH-1 consists of two domains: the peripheral arm (NuoB, -C, -D, -E, -F, -G, and -I) and the membrane arm (NuoA, -H, -J, -K, -L, -M, and -N). In Escherichia coli NDH-1, the hydrophilic subunits NuoC/Nqo5/30k and NuoD/Nqo4/49k are fused together in a single polypeptide as the NuoCD subunit. The NuoCD subunit is the only subunit that does not bear a cofactor in the peripheral arm. While some roles for inhibitor and quinone association have been reported for the NuoD segment, structural and functional roles of the NuoC segment remain mostly elusive. In this work, 14 highly conserved residues of the NuoC segment were mutated and 21 mutants were constructed using the chromosomal gene manipulation technique. From the enzymatic assays and immunochemical and blue-native gel analyses, it was found that residues Glu-138, Glu-140, and Asp-143 that are thought to be in the third α-helix are absolutely required for the energy-transducing NDH-1 activities and the assembly of the whole enzyme. Together with available information for the hydrophobic subunits, we propose that Glu-138, Glu-140, and Asp-143 of the NuoC segment may have a pivotal role in the structural stability of NDH-1.     

Characterization of the NuoM (ND4) subunit in Escherichia coli NDH-1: conserved charged residues essential for energy-coupled activities

The proton-translocating NADH-quinone (Q) oxidoreductase (NDH-1) from Escherichia coli is composed of two segments: a peripheral arm and a membrane arm. The membrane arm contains 7 hydrophobic subunits. Of these subunits, NuoM, a homolog of the mitochondrial ND4 subunit, is proposed to be involved in proton translocation and Q-binding. Therefore, we conducted site-directed mutation of 15 amino acid residues of NuoM and investigated their properties. In all mutants, the assembly of the whole enzyme seemed intact. Mutation of highly conserved Glu144 and Lys234 leads to almost total elimination of energy-transducing NDH-1 activities as well as increased production of superoxide radicals. Their NADH dehydrogenase activities were almost normal. Because these two residues are predicted to be located in the transmembrane segments of NuoM, the results strongly suggest that they participate in proton translocation. Although it is hypothesized that His interacts with a Q head group, mutations at four His moderately inhibited NDH-1 activities and had almost no effect on the Km values for Q or IC50 values of capsaicin-40, a competitive inhibitor for the Q binding site. The data suggest that these His are not involved in the catalytic Q-binding. Functional roles of NuoM and advantages of NDH-1 research as a model for mitochondrial complex I study have been discussed.     

A pair of membrane-embedded acidic residues in the NuoK subunit of Escherichia coli NDH-1, a counterpart of the ND4L subunit of the mitochondrial complex I, are required for high ubiquinone reductase activity

The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices. We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis. The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined. Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed. The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E. coli cells on malate as the main carbon source. The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes. Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant.     

Functional roles of four conserved charged residues in the membrane domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The H(+)(Na(+))-translocating NADH-quinone (Q) oxidoreductase (NDH-1) of Escherichia coli is composed of 13 different subunits (NuoA-N). Subunit NuoA (ND3, Nqo7) is one of the seven membrane domain subunits that are considered to be involved in H(+)(Na(+)) translocation. We demonstrated that in the Paracoccus denitrificans NDH-1 subunit, Nqo7 (ND3) directly interacts with peripheral subunits Nqo6 (PSST) and Nqo4 (49 kDa) by using cross-linkers (Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388 and Kao, M.-C., Matsuno-Yagi, A., and Yagi, T. (2004) Biochemistry 43, 3750-3755). To investigate the structural and functional roles of conserved charged amino acid residues, a nuoA knock-out mutant and site-specific mutants K46A, E51A, D79N, D79A, E81Q, E81A, and D79N/E81Q were constructed by utilizing chromosomal DNA manipulation. In terms of immunochemical and NADH dehydrogenase activity-staining analyses, all site-specific mutants are similar to the wild type, suggesting that those NuoA site-specific mutations do not significantly affect the assembly of peripheral subunits in situ. In addition, site-specific mutants showed similar deamino-NADH-K(3)Fe(CN)(6) reductase activity to the wild type. The K46A mutation scarcely inhibited deamino-NADH-Q reductase activity. In contrast, E51A, D79A, D79N, E81A, and E81Q mutation partially suppressed deamino-NADH-Q reductase activity to 30, 90, 40, 40, and 50%, respectively. The double mutant D79N/E81Q almost completely lost the energy-transducing NDH-1 activities but did not display any loss of deamino-NADH-K(3)Fe(CN)(6) reductase activity. The possible functional roles of residues Asp-79 and Glu-81 were discussed.     

Modeling of human pathogenic mutations in Escherichia coli complex I reveals a sensitive region in the fourth inside loop of NuoH

Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations.Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K_m value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.     

Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF+E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe-4S] cluster (g(x,y,z)=1.90, 1.95, and 2.05) in NuoF, and a [2Fe-2S] cluster (g(x,y,z)=1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx2Cx2Cx40C398 motif.     

Amiloride inhibition of the proton-translocating NADH-quinone oxidoreductase of mammals and bacteria

The proton-translocating NADH-quinone oxidoreductase in mitochondria (complex I) and bacteria (NDH-1) was shown to be inhibited by amiloride derivatives that are known as specific inhibitors for Na(+)/H(+) exchangers. In bovine submitochondrial particles, the effective concentrations were about the same as those for the Na(+)/H(+) exchangers, whereas in bacterial membranes the inhibitory potencies were lower. These results together with our earlier observation that the amiloride analogues prevent labeling of the ND5 subunit of complex I with a fenpyroximate analogue suggest the involvement of ND5 in H(+) (Na(+)) translocation and no direct involvement of electron carriers in H(+) (Na(+)) translocation.     

Features of Subunit NuoM (ND4) in Escherichia coli NDH-1: TOPOLOGY AND IMPLICATION OF CONSERVED GLU144 FOR COUPLING SITE 1*

The bacterial H⁺-pumping NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzymatic complex. Escherichia coli NDH-1 is composed of 13 subunits (NuoA–N). NuoM (ND4) subunit is one of the hydrophobic subunits that constitute the membrane arm of NDH-1 and was predicted to bear 14 helices. We attempted to clarify the membrane topology of NuoM by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. From the data, we propose a topology model containing 12 helices (helices I–IX and XII–XIV) located in transmembrane position and two (helices X and XI) present in the cytoplasm. We reported previously that residue Glu¹⁴⁴ of NuoM was located in the membrane (helix V) and was essential for the energy-coupling activities of NDH-1 (Torres-Bacete, J., Nakamaru-Ogiso, E., Matsuno-Yagi, A., and Yagi, T. (2007) J. Biol. Chem. 282, 36914–36922). Using mutant E144A, we studied the effect of shifting the glutamate residue to all sites within helix V and three sites each in helix IV and VI on the function of NDH-1. Twenty double site-directed mutants including the mutation E144A were constructed and characterized. None of the mutants showed alteration in the detectable levels of expressed NuoM or on the NDH-1 assembly. In addition, most of the double mutants did not restore the energy transducing NDH-1 activities. Only two mutants E144A/F140E and E144A/L147E, one helix turn downstream and upstream restored the energy transducing activities of NDH-1. Based on these results, a role of Glu¹⁴⁴ for proton translocation has been discussed.     

Leber hereditary optic neuropathy mutations in the ND6 subunit of mitochondrial complex I affect ubiquinone reduction kinetics in a bacterial model of the enzyme

LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.     

Bacterial NADH-quinone oxidoreductases

The NADH-quinone oxidoreductases of the bacterial respiratory chain could be divided in two groups depending on whether they bear an energy-coupling site. Those enzymes that bear the coupling site are designated as NADH dehydrogenase 1 (NDH-1) and those that do not as NADH dehydrogenase 2 (NDH-2). All members of the NDH-1 group analyzed to date are multiple polypeptide enzymes and contain noncovalently bound FMN and iron-sulfur clusters as prosthetic groups. The NADH-ubiquinone-1 reductase activities of NDH-1 are inhibited by rotenone, capsaicin, and dicyclohexylcarbodiimide. The NDH-2 enzymes are generally single polypeptides and contain non-covalently bound FAD and no iron-sulfur clusters. The enzymatic activities of the NDH-2 are not affected by the above inhibitors for NDH-1. Recently, it has been found that both of these types of the NADH-quinone oxidoreductase are present in a single strain of bacteria. The significance of the occurrence of these two types of enzymes in a single organism has been discussed in this review.     

Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.     

Subunit proximity in the H+-translocating NADH-quinone oxidoreductase probed by zero-length cross-linking

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (designated Nqo1-14), seven of which are located in the membrane domain and the other seven in the peripheral domain. It has been previously reported that membrane domain subunit Nqo7 (ND3) directly interacts with peripheral subunit Nqo6 (PSST) by using a cross-linker, m-maleimidobenzoyl-N-hydrosuccinimide ester, and heterologous expression [Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388]. To further explore the near-neighbor relationship of the subunits, a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and the Paracoccus membranes were used, and the cross-linked products were examined with antibodies specific to subunits Nqo1-11. The Nqo6 subunit was cross-linked to subunit Nqo9 (TYKY). In addition, a ternary product of Nqo3 (75k), Nqo6, and Nqo7 and binary products of Nqo3 and Nqo6 and of Nqo6 and Nqo7 were observed, but a binary product of Nqo3 and Nqo7 was not detected. The Nqo4 (49k) subunit was found to be associated with the Nqo7 subunit. Furthermore, Paracoccus subunits Nqo3, Nqo6, and Nqo7 were heterologously coexpressed in Escherichia coli, and EDC cross-linking experiments were carried out using the E. coli membranes expressing these three subunits. The results were the same as those obtained with Paracoccus membranes. On the basis of the data, subunit arrangements of NDH-1 were discussed.     

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.     

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.     

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.     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

Characterization of the iron-sulfur cluster coordinated by a cysteine cluster motif (CXXCXXXCX27C) in the Nqo3 subunit in the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX(27)C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g(z, y, x) = 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g( parallel, perpendicular) = 2.045, approximately 1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX(27)C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] 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.