The Proton-translocating NADH-Quinone Oxidoreductase (NDH-1) of Thermophilic Bacterium Thermus thermophilus HB-8
COMPLETE DNA SEQUENCE OF THE GENE CLUSTER AND THERMOSTABLE PROPERTIES OF THE EXPRESSED NQO2 SUBUNIT*

(Received for publication, September 13, 1996, and in revised form, November 20, 1996)

Takahiro Yano Dagger , Samuel S. Chu Dagger , Vladimir D. Sled' §dagger , Tomoko Ohnishi § and Takao Yagi Dagger

From the Dagger  Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the § Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The genes encoding the proton-translocating NADH-quinone oxidoreductase (NDH-1) of a thermophilic bacterium Thermus thermophilus HB-8 were cloned and sequenced. They constitute a cluster that is composed of 14 structural genes and contains no unidentified reading frames. All of the 14 structural genes, which are designated NQO1-14, encode subunits homologous to those of Paracoccus denitrificans NDH-1, respectively, and are arranged in the same order as other bacterial NDH-1 genes. T. thermophilus NDH-1 contains at most nine putative iron-sulfur cluster binding sites, eight of which are commonly found in other organisms. The T. thermophilus NQO2 subunit was expressed in Escherichia coli. The expressed subunit bears a single [2Fe-2S] cluster whose optical and EPR properties are very similar to those of N1a cluster in the P. denitrificans NQO2 subunit (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). These results strongly suggest that the T. thermophilus NDH-1 is similar to other NDH-1 enzyme complexes in terms of subunit and cofactor composition. The T. thermophilus NQO2 subunit displayed much higher stability than the mesophilic equivalent and its iron-sulfur cluster remained intact even after incubation for 3 h at 65 °C under anaerobic conditions. With the advantage of thermostability, the T. thermophilus NDH-1 provides a great model system to investigate the structure-function relationship of the NDH-1 enzyme complexes.


INTRODUCTION

NADH-quinone oxidoreductase (Complex I)1 of the mitochondrial respiratory chain is a membrane-bound enzyme complex that catalyzes the oxidation of NADH with ubiquinone as an electron acceptor. This enzyme complex concomitantly pumps protons across the inner membrane to generate an electrochemical proton gradient by utilizing energy obtained from the redox reaction as a driving force. Mitochondrial Complex I has been extensively studied using the bovine heart enzyme (1, 2). Complex I is composed of at least 41 dissimilar subunits, and its molecular mass is estimated to be more than 900 kDa. Complex I contains one non-covalently bound FMN and several iron-sulfur clusters as redox components. Some of the latter are EPR-visible and are designated N1a and N1b (for binuclear clusters) and N2, N3, and N4 (for tetranuclear clusters).

Recently, bacterial proton-translocating NADH-quinone oxidoreductase (NDH-1) has emerged as a useful and simpler model system for the study of Site I energy coupling (3). NDH-1 enzyme complexes have been isolated and characterized from several bacteria such as Paracoccus denitrificans and Escherichia coli (4, 5). The bacterial NDH-1 is a multiple-subunit enzyme complex and contains one non-covalently bound FMN and several iron-sulfur clusters similar to the mitochondrial Complex I. Genes encoding the NDH-1 were first cloned and sequenced from P. denitrificans (6-10). The genes constitute a cluster that is composed of 14 structural genes (designated NQO1-14) and 6 unidentified reading frames (URFs). All of the 14 subunits have mitochondrial homologues; seven subunits correspond to promontory, nuclear encoded subunits, and seven correspond to the hydrophobic mitochondrially encoded subunits, the so-called ND gene products (11). The P. denitrificans NDH-1 conserves all putative cofactor binding sites, suggesting that mitochondrial Complex I and NDH-1 share a similar structure and functional mechanism (3, 12). Recently, complete DNA sequences of E. coli NDH-1 and partial sequences of Salmonella typhimurium, Rhodobacter capsulatus, and Synechocystis sp. PCC6803 NDH-1 have been reported (13-16). Those sequences have shown the same structural features, providing further support that the bacterial NDH-1 is akin to the mitochondrial Complex I.

Thermus thermophilus strain HB-8, which was isolated from a hot spring in Japan, is an extremely thermophilic, obligatory aerobic, Gram-negative, and chemoheterotrophic bacterium (17). The bacterium is capable of growing in the temperature range of 45-85 °C with optimal growth temperature of 70 °C. It was suggested that its respiratory chain may include energy coupling Site I (18). Later, it was shown that T. thermophilus HB-8 possesses two types of NADH-quinone oxidoreductase (19). One is a proton-translocating NADH-dehydrogenase (NDH-1), and the other is a small non-energy-coupled enzyme referred to as NDH-2. Both enzymes have been purified and characterized to some extent. The NDH-1 was found to be composed of at least 10 dissimilar polypeptides and contained one non-covalently bound FMN, 11-12 mol of non-heme iron and 7-9 mol of acid-labile sulfide/mol. The NDH-2 was shown to be a single polypeptide with non-covalently bound FAD and no iron-sulfur clusters. Both enzymes can utilize NADH and deamino-NADH as substrates (in contrast to mesophilic NDH-2) and catalyze NADH-oxidation with water-soluble electron acceptors such as K3[Fe(CN)6] and Q1 at high temperature (>= 80 °C) (19). More recently, iron-sulfur clusters of the membrane-bound T. thermophilus HB-8 NDH-1 enzyme complex have been studied by EPR spectroscopy. At least three iron-sulfur clusters (one binuclear and two tetranuclear clusters) and possibly two additional iron-sulfur clusters (one binuclear and one tetranuclear) with very low redox mid-point potentials were detected in NDH-1 (20). Notably, cluster N2 signals were not seen in the study. We expect that the thermostability will provide a great advantage for structural studies of the NDH-1 either by NMR or x-ray crystallography. Furthermore, since T. thermophilus contains only menaquinone, it will provide an interesting system for understanding the Site I energy coupling mechanism.

This paper reports molecular cloning, sequencing, and expression studies of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of T. thermophilus HB-8. The genes encoding the T. thermophilus NDH-1 enzyme complex constitute a gene cluster that is composed of 14 structural genes (designated NQO1-14)2 and no URFs. All of the 14 structural genes encode subunits homologous to the P. denitrificans NDH-1 enzyme complex. Comparison of the deduced amino acid sequences with those of other organisms revealed that T. thermophilus NDH-1 possesses principally the same molecular structure as other bacterial NDH-1 in terms not only of subunit composition but also of cofactor binding sites. The NQO2 subunit of T. thermophilus NDH-1 was expressed in E. coli, purified, and partially characterized.


EXPERIMENTAL PROCEDURES

Genomic DNA Preparation

T. thermophilus HB-8 (ATCC27634) was grown in Thermus nutrient medium at 70 °C to late exponential phase according to Hon-nami and Oshima (21). The cells were harvested by centrifugation in a SS34 rotor at 6,000 rpm for 10 min. The cells were washed once with 10 mM Tris-HCl (pH 8.0) containing 0.15 M NaCl and 0.1 mM EDTA and resuspended in 2.0 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM EDTA (TE buffer). The cell suspension was then treated with 3 mg/ml lysozyme at 42 °C for 20 min. Twelve ml of 0.1 M Tris-HCl (pH 8.0) containing 0.1 M NaCl and 1% (w/v) SDS were added, and the cell suspension was freeze-thawed three times with liquid nitrogen and a 60 °C water bath. DNA was extracted with 14 ml of phenol/chloroform/TE for 10 min, followed by centrifugation three times. The aqueous phase was carefully transferred and dialyzed against 1 liter of 50 mM Tris-HCl (pH 8.0) containing 10 mM NaCl overnight with one buffer exchange. The DNA solution was incubated with 100 µg/ml RNase A at 37 °C for 3 h and then with 100 µg/ml proteinase K and 0.1% (w/v) SDS at 50 °C for 2 h. The solution was successively treated with an equal volume of phenol/chloroform/TE and then with chloroform/isoamyl alcohol. DNA was collected with a glass rod after the addition of 2.5 volumes of cold ethanol and suspended in TE buffer.

Cloning and Sequencing of the T. thermophilus NDH-1 Genes

To date, two complete (P. denitrificans and E. coli) and three partial DNA sequences (S. typhimurium, R. capsulatus, and Synechocystis) of the NDH-1 enzyme complexes have been deduced and are available in GenBankTM (6-10, 13-16). In the two bacteria, the NDH-1 genes are organized as a gene cluster. Therefore, it could be anticipated that T. thermophilus NDH-1 genes also constitute a gene cluster. Since no amino acid sequence information was available in T. thermophilus NDH-1, we decided to amplify DNA fragments by polymerase chain reaction (PCR) with oligonucleotides designed on the basis of sequence similarities between organisms whose sequences had already been determined. The NADH-binding subunit of T. thermophilus NDH-1 was previously identified as the 47-kDa subunit by photoaffinity labeling with [32P]NAD(H), and its amino acid composition was found to be very similar to those of P. denitrificans NQO1 and bovine heart Complex I FP 51-kDa subunit (22). We selected the NADH-binding subunit as the target, since it is expected that the primary sequence of the 47-kDa subunit is well conserved. The following oligonucleotides were derived from amino acid sequences that were highly conserved among several organisms (P. denitrificans NQO1, bovine heart complex I FP 51 kDa subunit, N. crassa 51-kDa subunit, E. coli NUOF, and S. typhimurium NUOF): TT1F (sense), 5'-ATC TG(C/T) GG(A/G/C/T) GA(A/G) GA(A/G) AC-3' from I217CGEET222 (P. denitrificans numbering); TT2F (sense), 5'-AAG CC(A/G/C/T) CC(A/G/C/T) TTC CC(A/G/C/T) GC-3' from K238PPFPA243; TT3R (antisense), 5'-TC(C/G) C(G/T)(A/G) CA(A/G/C/T) GG(C/G) GT(A/G) CA-3' from C394TPCR(D/E)399. DNA was amplified in 50 µl of reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 200 µM each of dNTPs (dATP, dGTP, dCTP, and dTTP), 1.0 µM each of sense and antisense primers, 1.5 µg of T. thermophilus HB-8 genomic DNA, and 2.5 units of Taq polymerase (Life Technologies, Inc.). The amplification was performed in a thermocycler with a reaction program that consisted of one cycle of hot start (94 °C for 10 min, 60 °C for 5 min), 35 amplification cycles that were composed of denaturing (94 °C for 1 min), annealing (50 °C for 1.5 min), and elongation (72 °C for 2 min), and finally one cycle of termination (72 °C for 10 min). Amplified DNA was purified with QIAquick PCR purification kit (QIAGEN) and run on a 1.0% (w/v) agarose gel. The DNA fragments of desired molecular sizes were excised and purified from gel with QIAquick gel extraction kit (QIAGEN) and then subcloned into TA cloning vector based on pBluescript KS(+) (Stratagene) that had been prepared according to Papp et al. (23). When two sets of oligonucleotides, TT1F (sense)/TT3R (antisense) and TT2F (sense)/TT3R (antisense), were used as primers, DNA fragments with molecular sizes of 540 and 480 bp were specifically amplified by PCR, respectively (data not shown). The 540-bp fragment contained an identical sequence to that of the 480-bp DNA fragment as expected. When the putative amino acid sequence derived from 540-bp DNA fragment was compared with those of the NADH-binding subunits of other organisms, a high degree of sequence similarity was found. Therefore, we concluded that the amplified DNA fragments encoded a part of the T. thermophilus NDH-1 NADH-binding subunit and could be used as a probe to clone the gene cluster. The standard cloning techniques used were according to Sambrook et al. (24). Southern blotting and colony hybridization were performed with DNA probes non-radioactively labeled with Genius 1 DNA labeling and detection kit (Boehringer Mannheim) according to the manufacture's protocol. With the 540-bp DNA fragment as the first probe, a 5.5-kilobase pair HindIII fragment was cloned (designated as pTTH-1) from a mini library constructed from 5.0-6.0-kilobase pair HindIII fragments digested from genomic DNA of T. thermophilus HB-8. The clone contained genes that were homologous to the downstream half of the PdNQO4, the entire region of the PdNQO2, NQO1, and NQO3 and the upstream part of the PdNQO8 (Fig. 1). DNA encompassing the entire T. thermophilus NDH-1 gene cluster was subsequently obtained as five clones that were designated as pTTH1-5, respectively, as shown in Fig. 1. DNA sequencing was performed using the dideoxy-chain termination method with the Applied Biosystems dye terminator cycle sequencing kit and T3, T7, and synthetic unique oligonucleotide primer. The DNA sequence of total 14,886 bp (from SacII to BamHI in Fig. 1) and the predicted amino acid sequences were deposited in GenBankTM under an accession number of TAU52917.


Fig. 1. Gene map of the T. thermophilus NDH-1 gene cluster. The gene map of the pTTH-1-5 DNA fragments is illustrated. The open reading frames, NQO1-14 genes, encode the T. thermophilus NDH-1 subunits homologous to NQO1-14 subunits of the P. denitrificans NDH-1, respectively. The PCR amplified DNA fragment is shown by an open bar. Several restriction enzyme recognition sites are shown: B, BamHI; E, EcoRV; H, HindIII; K, KpnI (not unique); S, SphI; S*, SacII (not unique). The CODONPREFERENCE program was employed to predict open reading frames. The three boxes below represent the translational reading frames of the DNA that are indicated by bold bars. The scale on each ordinate represents the relative probability of coding.
[View Larger Version of this Image (35K GIF file)]


Sequence Analysis

GCG software programs were used to analyze the DNA and amino acid sequences (25). Sequence comparison of the polypeptides were conducted with BESTFIT and PILEUP programs. The FASTA and PROFILESEARCH programs were used to search the GenBankTM/EMBL sequence data bases for proteins having some homology to the polypeptides. Homology search was also carried out by using the BLAST program running at the National Center for Biotechnology Information. Coding regions were searched with CODONPREFERENCE, and hydrophobic profiles were examined with HYDROPLOT.

Construction of an Expression Vector for the T. thermophilus NQO2 (TthNQO2) Subunit

The NQO2 gene was obtained from the pTTH-1 plasmid by PCR using two oligonucleotides to introduce NdeI site and BglII restriction sites at the translation initiation site and near the stop codon, respectively: NQO2F (sense), 5'-GG<UNL><IT>C</IT></UNL> <UNL>A<IT>T</IT>A</UNL> <UNL>TG</UNL>G GGT TCT TTG CGA C-3'; NQO2R (antisense), 5'-AA<UNL>A</UNL> <UNL>GA<IT>T</IT></UNL> <UNL><IT>CT</IT></UNL>G GGC CAG TCA TAC CTC C-3' (The italic bases were altered from T. thermophilus DNA, and the underlined bases indicate the newly introduced restriction sites, NdeI site for NQO2F and BglII site for NQO2R). The amplification was performed as described previously. The amplified DNA fragment (560 bp) was purified by agarose gel electrophoresis and subcloned into the pBluescript KS(+) vector. The integrity of the entire NQO2 gene was verified by sequencing. The construct was digested with NdeI and BglII and ligated into NdeI and BamHI sites of the pET16b expression vector. The final construct was designated pET16b(TthNQO2) and was used for the expression study.

Expression and Purification of the TthNQO2 Subunit from E. coli

Expression of the TthNQO2 subunit was conducted according to Yano et al. (26). The cells were suspended in 50 mM Tris-HCl (pH 7.4) containing 300 mM NaCl, 1.0 mM DTT, 1.0 mM PMSF, and 20 µg/ml leupeptin. The cell suspensions were treated by freeze-thawing twice using liquid nitrogen and a 30 °C water bath and were then passed twice through a Parr cell disruption bomb (>1,000 kg/cm2). The resulting suspension was centrifuged in a SS34 rotor at 12,000 rpm for 10 min. The cell-free extracts thus obtained were degassed and purged with oxygen-free argon and then brought into an anaerobic chamber. Afterward, all procedures were performed under anaerobic conditions to prevent destruction of iron-sulfur clusters by oxygen. The cell-free extracts were centrifuged in a 60Ti rotor at 48,000 rpm for 60 min. The TthNQO2 subunit was purified from the cytoplasmic fraction following two different procedures. For Protocol 1, the cytoplasmic fractions were dialyzed against 1 liter of degassed 50 mM potassium phosphate buffer (pH 7.0) containing 1.0 mM PMSF, and 20 µg/ml leupeptin for 6 h and then applied onto a nickel chelation column (1.0 × 1.5 cm) equilibrated with the same buffer. The column was washed with 50 ml of degassed 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 M NaCl, 1.0 mM PMSF, 20 µg/ml leupeptin, and 20 µg/ml pepstatin A, 80 mM imidazole, 10% (v/v) glycerol, and 0.1% (w/v) Tween 20, 50 ml of the same buffer without glycerol and Tween 20, and then the adsorbed TthNQO2 subunit was eluted with degassed 50 mM potassium phosphate buffer (pH 7.0) containing 0.5 M NaCl, 1.0 mM PMSF, and 400 mM imidazole. The purified subunit was dialyzed against 1 liter of 50 mM potassium phosphate buffer (pH 7.0) containing 1.0 mM DTT, and 0.1 mM PMSF overnight. For Protocol 2, the cytoplasmic fractions were incubated at 65 °C for 60 min under an anaerobic condition and chilled on ice for 10 min. The resultant solutions were then centrifuged in a SS34 rotor at 12,000 rpm for 15 min to remove denatured proteins. The transparent supernatants thus obtained were dialyzed against 1 liter of degassed 50 mM potassium phosphate buffer (pH 7.0) containing 1.0 mM PMSF, and 20 µg/ml leupeptin for 6 h and then applied onto a nickel chelation column (1.0 × 1.5 cm). The TthNQO2 subunit was purified in the same way as Protocol 1.

Preparation of T. thermophilus HB-8 Membranes

T. thermophilus HB-8 membranes were prepared according to Yagi et al. (19). Cells were grown at 75 °C in Thermus nutrient medium to late exponential phase according to Hon-nami and Oshima (21). Cells were harvested by centrifugation and suspended in 10 mM Tris-SO4 buffer (pH 7.5) containing 10 mM MgSO4 and 1.0 mM EDTA. The cell suspension was passed through a Parr cell disruption bomb (>1,000 kg/cm2) twice. The resultant suspension was centrifuged at 30,000 × g for 90 min. The pellet was homogenized with the same buffer and centrifuged. The pellet was resuspended in the same buffer. The membrane fractions were frozen in liquid nitrogen and stored at -80 °C until used.

EPR Measurements

EPR spectra were recorded by a Bruker ESP300 E spectrometer operating in the X band (9.2 GHz). The sample temperature was varied using an Oxford instrument ESR-9 helium flow cryostat. The magnetic field was calibrated using a strong or weak pitch standard. Spin quantitations were performed under non-power-saturated conditions using 500 µM Cu-EDTA as a standard.

Other Analytical Procedures

UV-visible absorption spectra were recorded on a SLM-Aminco DW-2000 spectrophotometer at room temperature. Protein concentration was estimated by the method of Lowry et al. (27) or by Biuret in the presence of 1 mg/ml sodium deoxycholate (28). SDS-polyacrylamide gel electrophoresis was carried out by a modified method of Laemmli (29). Immunoblotting was conducted as described previously (30-32). Non-heme iron and acid-labile sulfide were determined according to Doeg and Ziegler (33) and Fogo and Popowski (34), respectively. DNA sequences were determined according to Sanger et al. (35). 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, and Coomassie Brilliant Blue R-250 were from Bio-Rad; DTT, and horseradish peroxidase-conjugating affinity-purified antibodies to rabbit IgG were from Calbiochem; expression vectors, pET16b, and E. coli strain BL21(DE3)pLysS were from Novagen. All other chemicals were of the highest grade available from Sigma.


RESULTS

Structural Properties of the Gene Cluster

One of the unusual features of the T. thermophilus NDH-1 genes is their very high G+C content (an average of 68.3%). High preference of G+C in the third codon position (93.9%) results in a unique codon usage (17, 36). In order to identify the reading frames of the cloned DNA, we utilized (i) a codon preference table that had been built on the basis of 45 T. thermophilus DNA coding sequences reported in GenBankTM, (ii) amino acid sequence similarities to homologous subunits of other organisms (human, bovine heart, N. crassa, P. denitrificans, E. coli, S. typhimurium, R. capsulatus, etc.), and (iii) relative distance of putative translation initiation sites to ribosome binding site (Shine-Dalgarno sequence) (37). It was found that the cluster is composed of 14 open reading frames and contains no URFs (Fig. 1 and Table I). Five structural genes utilize unique translation start codons. GTG start codons were predicted in NQO6, NQO8, NQO11, and NQO13, and TTG start codon in the NQO7 gene (Table I). To our knowledge, nine genes have been found to utilize GTG start codon out of 45 T. thermophilus DNA sequences in GenBankTM. However, no TTG start codon has been reported to date in T. thermophilus. These rare start codons should be considered as tentative, and the actual translation start positions of the open reading frames must be experimentally identified in the future. The NDH-1 gene cluster contains a promoter-like sequence in its upstream region. The sequence 5'-CCCCTTGCG-3' and 5'-TAAGAT-3' are homologous to those of promoter regions of other T. thermophilus genes that have been empirically studied (38). These sequences are tentatively assigned to be -35 and -10 boxes, respectively. The gene cluster ends with a stem-loop-like structure that starts 49 bp downstream of the NQO14 stop codon. In addition, all 14 structural genes are so compactly organized in the cluster that there is almost no intergenic space where promoter or terminator-like sequences can be present. These properties strongly suggest that the T. thermophilus NDH-1 genes constitute an operon.

Table I.

Characteristics of T. thermophilus HB-8 NDH-1 gene cluster


Gene DNA length G + C content Initiation codon Termination codon No. of amino acid residues Mr pI P. denitrificans homologue Bovine homologue

bp %
NQO1 1,317 67.4 ATG TGA 438 48,631.4 7.48 NQO1 51-kDa
NQO2 546 66.3 ATG TGA 181 20,286.1 4.85 NQO2 24-kDa
NQO3 2,352 70.2 ATG TGA 783 86,558.2 6.99 NQO3 75-kDa
NQO4 1,230 66.0 ATG TAA 409 46,370.8 5.33 NQO4 49-kDa
NQO5 624 66.8 ATG TGA 207 23,859.0 5.89 NQO5 30-kDa
NQO6 546 67.6 GTG TGA 181 20,238.4 9.39 NQO6 PSST
NQO7 360 65.6 TTG TGA 119 13,144.7 9.13 NQO7 ND3
NQO8 1,098 65.5 GTG TGA 365 41,008.3 9.53 NQO8 ND1
NQO9 549 67.9 ATG TGA 182 20,080.1 7.53 NQO9 TYKY
NQO10 531 68.9 ATG TGA 176 18,551.2 5.91 NQO10 ND6
NQO11 288 69.4 GTG TAA 95 9,995.7 5.78 NQO11 ND4L
NQO12 1,821 68.0 ATG TAG 606 65,140.6 9.79 NQO12 ND5
NQO13 1,410 68.9 GTG TGA 469 49,393.9 6.10 NQO13 ND4
NQO14 1,296 72.2 ATG TGA 431 44,964.8 10.53 NQO14 ND2
Total 68.3 4,642 508,223.3

Predicted Amino Acid Sequence of Individual Subunits of the T. thermophilus NDH-1

The deduced primary sequences of the T. thermophilus NDH-1 subunits were compared with those of other organisms (e.g. bovine heart, N. crassa, P. denitrificans, and E. coli) and with those of some phylogenetically related enzymes (e.g. Alcaligenes eutrophus NAD+-reducing hydrogenase, E. coli formate hydrogen lyase). All of the 14 subunits of the T. thermophilus NDH-1 are homologous to their mitochondrial and bacterial counterparts. Here, several important and interesting features of individual subunits are described in conjunction with the latest information of mitochondrial Complex I and bacterial NDH-1.

TthNQO1 Subunit

The TthNQO1 subunit is homologous to bovine FP 51-kDa, PdNQO1, and EcNuoF subunits (Fig. 2 and Table II). This subunit plays an essential role in initiating the redox reaction by oxidizing NADH. As described above, the 47-kDa subunit was photoaffinity-labeled by [32P]NAD(H), indicating that the NADH-binding site is located in the subunit (22). The TthNQO1 subunit conserves a typical nucleotide binding sequence motif (G64XG66XXG69) with remote acidic amino acid residues D94E95. A tetranuclear iron-sulfur cluster (N3) is thought to be located in the NADH-binding subunits (39, 40). The TthNQO1 subunit contains five conserved cysteine residues (Cys182, Cys353, Cys356, Cys359, and Cys400). Four of them form a cluster with a typical motif for a [4Fe-4S] cluster binding site (C353XXC356XXC359), and Cys400 is followed by Pro (Fig. 2). Therefore, Cys400 seems more likely to be the fourth ligand residue of the [4Fe-4S] cluster than Cys182. The TthNQO1 subunit and its homologues are also thought to accommodate an FMN that is an indispensable component for NADH oxidation (40). As discussed in previous papers (2, 6, 41), however, it is difficult to predict the FMN-binding site on the basis of sequence data at the present time mainly due to lack of consensus sequence motifs for this cofactor.


Fig. 2. Comparison of the amino acid sequences of the T. thermophilus NQO1 subunit with its homologues from various organisms. The comparison was conducted using the PILEUP program. Bovine, bovine heart mitochondrial Complex I; P.d., P. denitrificans; T.th., T. thermophilus; HoxU, A. eutrophus NAD+-reducing hydrogenase gamma  subunit. The putative nucleotide binding sites are indicated by #. * indicates the conserved cysteine residues. An arrow (Up-arrow ) indicates a proline residue that is unique to the T. thermophilus NQO1 subunit. Boxed sequences correspond to those used to synthesize the PCR primers TT1F, TT2F, and TT3R (indicated by arrows: right-arrow, left-arrow ).
[View Larger Version of this Image (70K GIF file)]


Table II.

Amino acid sequence identities of T. thermophilus NDH-1 subunits to homologues of bovine mitochondrial Complex I, P. denitrificans NDH-1, and E. coli NDH-1


T. thermophilus NDH-1 vs. bovine Complex I vs. P. denitrificans NDH-1 vs. E. coli NDH-1

NQO1 46.0%  (FP 51-kDa)a 43.6%  (NQO1) 47.0%  (NUOF)
NQO2 33.7%  (FP 24-kDa) 34.3%  (NQO2) 28.4%  (NUOE)
NQO3 30.4%  (IP 75-kDa) 30.4%  (NQO3) 28.2%  (NUOG)
NQO4 44.4%  (IP 49-kDa) 42.6%  (NQO4) 40.6%  (NUOD)
NQO5 27.8%  (IP 30-kDa) 33.0%  (NQO5) 39.6%  (NUOC)
NQO6 52.8%  (PSST) 52.4%  (NQO6) 48.0%  (NUOB)
NQO7 30.6%  (ND3) 40.0%  (NQO7) 31.6%  (NUOA)
NQO8 42.2%  (ND1) 41.0%  (NQO8) 47.7%  (NUOH)
NQO9 42.1%  (TYKT) 39.2%  (NQO9) 45.0%  (NUOI)
NQO10 19.5%  (ND6) 31.4%  (NQO10) 31.6%  (NUOJ)
NQO11 30.4%  (ND4L) 44.1%  (NQO11) 33.7%  (NUOK)
NQO12 34.0%  (ND5) 44.8%  (NQO12) 45.3%  (NUOL)
NQO13 26.5%  (ND4) 34.0%  (NQO13) 30.7%  (NUOM)
NQO14 27.6%  (ND2) 35.4%  (NQO14) 39.4%  (NUON)

a  Respective counterparts are indicated in parentheses.

TthNQO2 Subunit

The TthNQO2 subunit is homologous to bovine FP 24-kDa, PdNQO2, and EcNuoE subunits (Table II). Expression studies of the PdNQO2 subunit have shown that this subunit bears a single [2Fe-2S] cluster, which is tentatively assigned as cluster N1a (26). Further mutagenesis studies of PdNQO2 subunit have indicated that the [2Fe-2S] cluster is coordinated by four conserved cysteine residues (Cys96, Cys101, Cys137, and Cys141, P. denitrificans numbering) (42). The TthNQO2 subunit also conserves these four cysteine residues (Cys83, Cys88, Cys124, and Cys128).

TthNQO3 Subunit

The TthNQO3 subunit is homologous to bovine IP 75-kDa, PdNQO3, and EcNuoG subunits (Fig. 3 and Table II). This subunit generally contains 12 invariant cysteine residues in the N-terminal region that are possible iron-sulfur cluster ligands (8, 14, 43). Recently, expression studies of the PdNQO3 subunit showed that the subunit contains multiple iron-sulfur clusters: one [2Fe-2S] cluster (N1b), one [4Fe-4S] cluster (N4), and possibly another [4Fe-4S] cluster (44). Interestingly, it was found that the TthNQO3 subunit conserves only 11 cysteine residues (Cys34, Cys45, Cys48, Cys83, Cys119, Cys122, Cys128, Cys181, Cys184, Cys187, and Cys230), as the 12th is replaced by Val23 (Fig. 3). It is worth noting that this cysteine residue is also absent from the HoxU subunit of A. eutrophus NAD+-reducing hydrogenase that contains the other 11 cysteine residues (Fig. 3) (45). Assuming three iron-sulfur clusters (one binuclear and two tetranuclear clusters) in the subunit, this implies that at least one of the clusters has to utilize a non-cysteinyl ligand residue. Currently, it is difficult to predict which iron-sulfur cluster utilizes a non-cysteinyl ligand residue. However, it can be anticipated that one of the [4Fe-4S] clusters is coordinated by four cysteine residues that form a typical sequence motif for a [4Fe-4S] cluster (C181XXC184XXC187 ... C230P231, T. thermophilus numbering). Although there is no typical binding sequence motifs for a binuclear iron-sulfur cluster, preliminary results of resonance Raman spectroscopic studies of the expressed PdNQO3 subunit have suggested that the [2Fe-2S] cluster (N1b) is coordinated by cysteinyl residues only.3 If another cluster, [4Fe-4S], is present, it must utilize a non-cysteinyl ligand within a novel binding motif. Furthermore, the TthNQO3 subunit contains an additional cysteine cluster (C256XXC259XXXC263 ... C291) that is also found in E. coli and S. typhimurium NuoG subunits but not in other NDH-1 enzyme complexes (Fig. 3). It has been reported that the E. coli NDH-1 enzyme contains an additional EPR detectable [2Fe-2S] cluster (three [2Fe-2S] clusters are designated as N1a, N1b, and N1c), although the exact location of the third [2Fe-2S] cluster is not known (5, 46). Thus, it is of interest to investigate if another [2Fe-2S] cluster is located in the TthNQO3 subunit and, if so, what role it plays in electron transfer. In order to address these issues, expression of the TthNQO3 subunit is in progress in our laboratory.


Fig. 3. Comparison of the amino acid sequences of the T. thermophilus NQO3 subunit with its homologues from various organisms. The comparison was conducted using the PILEUP program. Bovine, bovine heart mitochondrial Complex I; P.d., P. denitrificans; T.th., T. thermophilus. Conserved cysteine residues are indicated by *. # indicates cysteine residues that are predicted to ligate a [2Fe-2S] cluster. An arrow (Up-arrow ) indicates the cysteine residue that is not conserved in the T. thermophilus NQO3 subunit.
[View Larger Version of this Image (91K GIF file)]


TthNQO4 and TthNQO5 Subunits

The TthNQO4 and TthNQO5 subunits are homologous to the bovine IP 49-kDa, PdNQO4, and EcNuoC subunits and to bovine IP 30-kDa, PdNQO5, and EcNuoB subunits, respectively (Table II). Recently, the PdNQO4 and PdNQO5 subunits were natively and individually expressed as soluble proteins in E. coli, showing that neither subunit contains iron-sulfur clusters (47). The same conclusion could be drawn from the sequences of T. thermophilus subunits. The TthNQO4 subunit contains only one non-conserved cysteine residue (Cys385), whereas the TthNQO5 subunit does not contain cysteine residues. The functional roles of these subunits are not known.

TthNQO6 Subunit

Among the 14 subunits, the TthNQO6 subunit showed the highest sequence identities to its bovine, P. denitrificans, and E. coli equivalents (Fig. 4A and Table II). These subunits are homologous to the HycG subunit of E. coli formate hydrogen lyase. This subunit contains conserved cysteine residues that are also present in TthNQO6 subunit (Cys45, Cys46, Cys111, and Cys140). The cysteine arrangement in the NQO6 subunit and its homologues has been suggested to be analogous to that in the small subunit of nickel-iron hydrogenases (48, 49). In the latter enzymes, involvement of the corresponding cysteines in ligating a [4Fe-4S] cluster has recently been revealed by the three-dimensional crystal structure of Desulfovibrio gigas hydrogenase (50). It remains to be determined whether the NQO6 subunit contains an iron-sulfur cluster.


Fig. 4. Comparison of the amino acid sequences of the T. thermophilus NQO6 (A) and NQO9 (B) subunits with their homologues from various organisms. The comparison was conducted using the PILEUP program. Bovine, bovine heart mitochondrial Complex I; P.d., P. denitrificans; R.c., R. capsulatus; T.th., T. thermophilus. The conserved cysteine residues are indicated by *.
[View Larger Version of this Image (59K GIF file)]


TthNQO9 Subunit

The TthNQO9 subunit is homologous to the bovine TYKY, PdNQO9, and EcNuoI subunits (Fig. 4B and Table II). Sequence suggests that this subunit contains iron-sulfur clusters. The TthNQO9 subunits contains 8 invariant cysteine residues (C53XXC56XXC59 ... C63P64, C98XXC101XXC104 ... C108P109) that are typical sequence motifs of [4Fe-4S] cluster binding sites as seen in 2×[4Fe-4S] ferredoxins (51, 52). Our preliminary results from an expression and reconstitution study of the PdNQO9 subunit have indicated that NQO9 indeed contains two [4Fe-4S] clusters.4 Iron-sulfur cluster N2, which probably plays a key role in the electron transfer pathway as a direct electron donor to quinone, has not yet been assigned to a subunit. The hypothetical cluster in NQO6 and either of those in NQO9 are possible candidates.

TthNQO7, TthNQO8, and TthNQO10-14 Subunits

T. thermophilus NDH-1 gene cluster contains seven genes (NQO7, NQO8, and NQO10-14) that encode subunits homologous to bovine mitochondrial ND3, ND1, ND6, ND4L, ND5, ND4, and ND2 gene products and to the other seven subunits of bacterial NDH-1 (Table II). These subunits are extremely hydrophobic and are thought to constitute the integral membrane part of the enzyme complex. The T. thermophilus subunits contain a number of hydrophobic stretches that are predicted to span the cytoplasmic membrane (Fig. 5). As has been suggested previously (1, 2, 5, 53, 54), the membrane assembly probably plays an essential role in quinone reduction and proton translocation. The T. thermophilus membrane subunits conserve several charged amino acid residues, some of which may be involved in the energy-tranducing mechanism. It is very difficult, however, to discuss specific functional roles of the individual subunits based only on the sequence data. Little is known about the structures of these subunits, and quinone-binding site(s) have not been identified. Thus, much future work is needed before any conclusions can be made.


Fig. 5. Hydropathy profiles of the T. thermophilus NQO7, NQO8, and NQO10-14 subunits. The hydrophobicity was analyzed using the HYDROPLOT program that is based on Kyte and Doolittle (66) with a window of 11 residues.
[View Larger Version of this Image (26K GIF file)]


Expression of the T. thermophilus NQO2 (TthNQO2) Subunit in E. coli

As mentioned previously, the TthNQO2 subunit contains the four conserved cysteine residues that were shown to ligate a [2Fe-2S] cluster in PdNQO2. The PdNQO2 subunit is one of the well characterized subunits in NDH-1 (26, 42, 55) and has similar physicochemical properties to Clostridium pasteurianum binuclear ferredoxin (56). Therefore, to explore the thermostable properties, we attempted to express the TthNQO2 subunit in E. coli and purify it. Its physicochemical properties could be directly compared with those of the PdNQO2 subunit. The TthNQO2 subunit was expressed as a soluble protein and could be purified with nickel chelation column chromatography (Fig. 6A). We raised polyclonal antibodies against the expressed TthNQO2 subunit and utilized them to examine the identity of the subunit with the T. thermophilus membrane preparation. The antibodies recognized a single polypeptide with an apparent molecular weight of Mr = 21,000 (Fig. 6B). These results indicate that the sequences were precisely analyzed and that the subunit was correctly expressed in E. coli. The purified TthNQO2 subunit was reddish-brown in color as previously observed with the PdNQO2 subunit. Its absorption spectrum showed great resemblance to that of the PdNQO2 subunit. Characteristic absorption peaks of the iron-sulfur cluster (330, 420, 460, and 550 nm) could be seen in its oxidized form (Fig. 7A). EPR measurements provided further evidence that the TthNQO2 subunit contains a single [2Fe-2S] cluster with similar properties to the PdNQO2 subunit (Fig. 7B).


Fig. 6. SDS-polyacrylamide gel pattern of the expressed TthNQO2 subunit in E. coli (A) and immunoblotting of T. thermophilus HB-8 membranes with anti-TthNQO2 subunit antibody (B). Panel A, the lanes contain cell lysate of E. coli harboring pET16b(TthNQO2) (10 µg of protein for lane 1), soluble fraction (8.0 µg of protein for lane 2), and the purified TthNQO2 subunit (1.0 µg of protein for lane 3). The molecular sizes are indicated on the left. Panel B, 30 µg of T. thermophilus HB-8 membrane protein were loaded on a 15% SDS-polyacrylamide gel. After electrophoresis the proteins were transferred onto a nitrocellulose membrane. Immunoblotting was carried out using antibodies against the expressed TthNQO2 subunit and horseradish peroxidase-conjugated anti-rabbit IgG antibody as described in Refs. 30-32, except that the detection was carried out using the ECL kit (Amersham). The molecular sizes are indicated on the left.
[View Larger Version of this Image (41K GIF file)]



Fig. 7. Absorption and EPR spectra of the expressed and purified TthNQO2 subunit. Panel A, the purified TthNQO2 subunit was diluted to 0.28 mg/ml in 10 mM potassium phosphate buffer (pH 7.0) containing 1.0 mM DTT. Absorption spectra of the expressed TthNQO2 subunit in its oxidized form (line 1) and in the 10 mM sodium dithionite reduced form (line 2) were recorded at room temperature. The difference spectrum (oxidized form - reduced form, 1-2) is shown in the inset. Panel B, EPR spectra of dithionite reduced [2Fe-2S] clusters in overexpressed purified TthNQO2 (line 1) and PdNQO2 (line 2) subunit. EPR conditions: microwave frequency, 9.519 GHz; microwave power, 2 milliwatts; modulation amplitude, 1 millitesla; modulation frequency, 100 kHz; time constant, 64 ms; sample temperature, 30 K. Arrows indicate principal g values of the cluster.
[View Larger Version of this Image (18K GIF file)]


Thermostability of the TthNQO2 Subunit

It has been reported that the isolated T. thermophilus NDH-1 enzyme complex exhibits extremely thermostable properties and is capable of catalyzing NADH oxidation at temperatures over 65 °C. In a preliminary experiment, the thermostability of the expressed TthNQO2 subunit was tested by incubating crude preparations (cytoplasmic fraction) at 65 °C for various time under anaerobic conditions. The solutions were centrifuged to remove denatured proteins, and the resultant supernatants were subjected to SDS-PAGE. As shown in Fig. 8A, while E. coli proteins were readily denatured by the heat treatment, the TthNQO2 subunit remained soluble in the supernatant. These results show that the TthNQO2 subunit is more heat-resistant than E. coli proteins. After subsequent purification by nickel chelation column chromatography, the subunit exhibited the characteristic red-brown color, indicating that the [2Fe-2S] cluster also survived the treatment (data not shown). The TthNQO2 subunit was further used in order to assess the degree of thermostability. The purified TthNQO2 subunit (by Protocol 1) was incubated at various temperatures (50-90 °C) for 30 min under anaerobic conditions followed by centrifugation at 14,000 rpm for 10 min. Absorption spectra of the supernatants were measured in the visible region. Almost no precipitation was observed up to 65 °C, and the absorption spectra of the subunits were unaltered (Fig. 8B, line 1). Notably, no spectral changes occurred at 65 °C during incubation up to 3 h (data not shown). When the temperature was elevated to 75-80 °C, the subunit started to show some instability such that some portion of the protein was denatured and precipitated (Fig. 8B, lines 2 and 3). At 85 °C and above, the denaturing process was further accelerated. The absorption spectrum of the soluble subunits was significantly modified (Fig. 8B, line 4). On the other hand, when the PdNQO2 subunit was heat-treated in the same way, the protein was immediately denatured even at 30 °C (not shown). These results clearly demonstrate that the T. thermophilus NDH-1 subunits are extremely stable and thus more suitable to express and study as single proteins than are their mesophilic equivalents. It should be noted that the iron-sulfur cluster in the TthNQO2 subunit was stable only under oxygen-free conditions. In the presence of oxygen, the subunit readily lost its reddish-brown color, indicating that the iron-sulfur cluster is vulnerable to attack by oxygen. This is often seen also in other iron-sulfur proteins (57).


Fig. 8. Thermostability measurement of the expressed TthNQO2 subunit. Panel A, the cytoplasmic fractions (~20-25 mg/ml) prepared from E. coli harboring pET16b(TthNQO2) in 50 mM Tris-HCl buffer (pH 7.4) containing 300 mM NaCl, 1.0 mM DTT, 1.0 mM PMSF, and 20 µg/ml leupeptin in 1.5-ml microtubes were incubated at 65 °C for various times (0, 5, 10, 15, 30, and 60 min, lanes 1-6, respectively) under an anaerobic condition and then chilled on ice for 10 min. The preparations were centrifuged at 14,000 rpm for 15 min to remove denatured proteins, and the transparent supernatants were transferred into new microtubes. The supernatants were mixed with an equal volume of SDS-PAGE sample buffer that contained 160 mM Tris-HCl (pH 6.8) and 12% (w/v) SDS, boiled for 10 min, and then subjected to 14% (w/v) SDS-polyacrylamide gel electrophoresis. Lane 7 represents a purified TthNQO2 subunit (1.0 µg). Panel B, 700 µl of the purified TthNQO2 subunit (0.25 mg/ml) in microtubes were incubated for 30 min at various temperatures (50-90 °C) and then chilled on ice for 10 min. After the solutions were centrifuged at 14,000 rpm for 15 min, the absorption spectra of the supernatants were recorded at room temperature. Lines 1, 2, 3, and 4 represent the absorption spectra of the subunit treated at 65, 75, 80, and 85 °C, respectively.
[View Larger Version of this Image (37K GIF file)]



DISCUSSION

The present study has revealed that the T. thermophilus NDH-1 (eukaryotic Complex I equivalent) genes constitute an operon-like structure that is composed of 14 structural genes (NQO1-14) with no URFs. The T. thermophilus NDH-1 is composed of seven predicted transmembranous subunits (NQO7, NQO8, and NQO10-14) and seven peripherally located subunits (NQO1-6 and NQO9), all of which are very homologous to the corresponding subunits of mitochondrial Complex I and other bacterial NDH-1. The order of the 14 structural genes is identical to those of other NDH-1 gene clusters (P. denitrificans and E. coli) and similar to those encoding other related enzymes, suggesting a phylogenetic relationship between bacterial NDH-1, NAD-reducing hydrogenase, and some related enzymes. These results further substantiate that the 14-subunit structure is the minimal and ubiquitous functional unit of Complex I and NDH-1.

Previous biochemical studies of the isolated T. thermophilus NDH-1 have shown that the enzyme contains one FMN and several iron-sulfur clusters as redox components (19). The T. thermophilus NDH-1 contains only 40 cysteine residues (while the P. denitrificans NDH-1 contains 64 cysteine residues), all of which are located in the extrinsic subunits (Table III). Thirty-two out of 40 cysteine residues are fully conserved among mitochondrial and bacterial Complex I whose amino acid sequences have been reported. As described under "Results," the T. thermophilus NDH-1 has the potential of bearing eight iron-sulfur clusters in common with mitochondrial and bacterial enzyme complexes and probably an additional [2Fe-2S] cluster in the TthNQO3 subunit, which is unique to T. thermophilus, E. coli, and S. typhimurium. It has been shown by EPR studies that at least three NADH-reducible iron-sulfur clusters (one binuclear and two tetranuclear) and possibly two iron-sulfur clusters (one binuclear and one tetranuclear) with very low redox midpoint potentials are present in the membrane-bound T. thermophilus NDH-1 (20). Although exact assignment of these EPR-detectable iron-sulfur clusters remains to be made, these results together with the sequence information in the present study indicate that the T. thermophilus NDH-1 possesses at least as many iron-sulfur clusters as its mitochondrial and bacterial equivalents.

Table III.

Distribution of the conserved cysteine residues and the putative iron-sulfur cluster binding sites in the T. thermophilus NDH-1


Subunits No. of cysteine residues Conserved cysteine residues Iron-sulfur clusters

NQO1 6  (5)a Cys182, Cys353, Cys356, Cys359, Cys400 [4Fe-4S] (N3)
NQO2 6  (4)a Cys83, Cys88, Cys124, Cys128 [2Fe-2S] (N1a)
NQO3 15  (11)a Cys34, Cys45, Cys48, Cys83, Cys119, Cys122, Cys128, Cys181, Cys184, Cys187, Cys230, (Cys256, [2Fe-2S] (N1b)
  Cys259, Cys263, Cys291)b [4Fe-4S] (N4)
[4Fe-4S] (?)
[2Fe-2S] (N1c?)
NQO6 4  (4)a Cys45, Cys46, Cys111, Cys140 [4Fe-4S] (N2?)
NQO9 8  (8)a Cys53, Cys56, Cys59, Cys63, Cys98, Cys101, Cys104, Cys108 2 × [4Fe-4S] (N2?)
Total 40c  (32)a 8-9 iron-sulfur clusters

a  Number of the cysteine residues strictly conserved among mitochondrial Complex I and bacterial NDH-1.
b  These cysteine residues are conserved only among T. thermophilus, E. coli, and S. typhimurium NDH-1.
c  One non-conserved cysteine residue is located in TthNQO4 subunit.

However, T. thermophilus NDH-1 seems to be unique among the known Site I enzyme complexes from a bioenergetic point of view, because T. thermophilus contains only menaquinone-8 (58). Menaquinone-8 has a lower redox midpoint potential than ubiquinone (Em,7 -75 mV for menaquinone/menaquinol couple; Em,7 = +100 mV for ubiquinone/ubiquinol couple). Thus, the free energy available from electron transfer from NADH to menaquinone is much less than when using ubiquinone. Therefore, comparative studies of T. thermophilus NDH-1 with ubiquinone-utilizing complexes may provide new insights into Site I energy coupling mechanism.

Thermostability of proteins isolated from not only T. thermophilus but also from several other thermophilic microorganisms has recently received considerable attention. Several attempts have been made to clarify the origin of the thermostability, and some principles have been proposed (59-61). Based on the comparative structural studies of thermophilic and mesophilic proteins, it seems likely that structural stability of thermophilic proteins are reinforced by an increment of intrapolypeptide interactions throughout the protein molecule (62-64). The present study has shown that the expressed TthNQO2 subunit is thermostable, since the subunit and its iron-sulfur cluster survived at 65 °C for 3 h. The same observation has been made for other T. thermophilus NDH-1 subunits and subcomplexes expressed in E. coli.5 These results indicate that heat resistance resides in individual subunits. The purified NQO2 subunit and other expressed subunits remained stable at room temperature (22-25 °C) for weeks in an anaerobic chamber without loosing the iron-sulfur cluster. The availability of these stable subunits give us the possibility to investigate subunit-subunit interaction by reconstituting the entire enzyme complex from individual components as demonstrated for ATPase (65). Furthermore, significant stability is also a great advantage for structural studies of the complex using x-ray crystallography or NMR spectroscopy.


FOOTNOTES

*   This work was supported by United States Public Health Service Grants R01GM33712 (to T. Y.) and R01GM30376 (to T. O.). Computer facilities were supported by United States Public Health Service Grant M01RR00833 for the General Clinical Research Center. Synthetic oligonucleotides and DNA sequencing were supported in part by the Sam & Rose Stein Endowment Fund. This is Publication 10232-MEM from The Scripps Research Institute, La Jolla, CA. 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.

This paper is dedicated to memory of Vladimir D. Sled'.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) TAU52917[GenBank].


dagger    Deceased.
   To whom reprint requests should be addressed: SBR-15, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Fax: 619-784-2054; E-mail: yagi{at}scripps.edu.
1    The abbreviations used are: Complex I, mitochondrial proton-translocating NADH-ubiquinone oxidoreductase; NDH-1, bacterial proton-translocating NADH-quinone oxidoreductase; NDH-2, bacterial NADH-quinone oxidoreductase lacking an energy coupling site; FP, flavoprotein fraction; IP, iron-sulfur protein fraction; URF, unidentified reading frame; Tth, T. thermophilus; Pd, P. denitrificans; Ec, E. coli; PCR, polymerase chain reaction; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis.
2    T. thermophilus contains only menaquinone. Since <UNL>N</UNL>ADH-<UNL>q</UNL>uinone <UNL>o</UNL>xidoreductase (NQO) refers to Complex I and NDH-1 interacting with any quinone species, we have decided to keep the nomenclature used for the P. denitrificans NQO subunits.
3    C. Staple, T. Yano, T. Yagi, and M. Johnson, unpublished results.
4    T. Yano, V. D. Sled', T. Ohnishi, and T. Yagi, unpublished results.
5    T. Yano and T. Yagi, unpublished data.

Acknowledgments

We thank Julieann Grant for excellent technical assistance; Drs. Saeko Takano, Tomomi Kitajima-Ihara, and Akemi Matsuno-Yagi for stimulating discussion; and Drs. Youssef Hatefi and Cecilia Hägerhäll for critical reading of the manuscript.


REFERENCES

  1. Hatefi, Y. (1985) Annu. Rev. Biochem. 54, 1015-1069 [CrossRef][Medline] [Order article via Infotrieve]
  2. Walker, J. E. (1992) Q. Rev. Biophys. 25, 253-324 [Medline] [Order article via Infotrieve]
  3. Yagi, T. (1993) Biochim. Biophys. Acta 1141, 1-17 [Medline] [Order article via Infotrieve]
  4. Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311 [Medline] [Order article via Infotrieve]
  5. Leif, H., Sled', V. D., Ohnishi, T., Weiss, H., and Friedrich, T. (1995) Eur. J. Biochem. 230, 538-548 [Abstract]
  6. Xu, X., Matsuno-Yagi, A., and Yagi, T. (1991) Biochemistry 30, 6422-6428 [Medline] [Order article via Infotrieve]
  7. Xu, X., Matsuno-Yagi, A., and Yagi, T. (1991) Biochemistry 30, 8678-8684 [Medline] [Order article via Infotrieve]
  8. Xu, X., Matsuno-Yagi, A., and Yagi, T. (1992) Arch. Biochem. Biophys. 296, 40-48 [Medline] [Order article via Infotrieve]
  9. Xu, X., Matsuno-Yagi, A., and Yagi, T. (1992) Biochemistry 31, 6925-6932 [Medline] [Order article via Infotrieve]
  10. Xu, X., Matsuno-Yagi, A., and Yagi, T. (1993) Biochemistry 32, 968-981 [Medline] [Order article via Infotrieve]
  11. Yagi, T., Yano, T., and Matsuno-Yagi, A. (1993) J. Bioenerg. Biomembr. 25, 339-345 [Medline] [Order article via Infotrieve]
  12. Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1993) Biol. Chem. Hoppe-Seyler 374, 820
  13. Steinmüller, K. (1992) Plant Mol. Biol. 18, 135-137 [Medline] [Order article via Infotrieve]
  14. Weidner, U., Geier, S., Ptock, A., Friedrich, T., Leif, H., and Weiss, H. (1993) J. Mol. Biol. 233, 109-122 [CrossRef][Medline] [Order article via Infotrieve]
  15. Archer, C. D., Wang, X., and Elliott, T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9877-9881 [Abstract]
  16. Dupuis, A., Peinnequin, A., Chevallet, M., Lunardi, J., Darrouzet, E., Pierrard, B., Procaccio, V., and Issartel, J. P. (1995) Gene (Amst.) 167, 99-104 [CrossRef][Medline] [Order article via Infotrieve]
  17. Oshima, T., and Imahori, K. (1974) Int. J. Syst. Bacteriol. 24, 102-112
  18. Mckay, A., Quilter, J., and Jones, C. W. (1982) Arch. Microbiol. 131, 43-50
  19. Yagi, T., Hon-nami, K., and Ohnishi, T. (1988) Biochemistry 27, 2008-2013 [Medline] [Order article via Infotrieve]
  20. Meinhardt, S. W., Wang, D.-C., Hon-nami, K., Yagi, T., Oshima, T., and Ohnishi, T. (1990) J. Biol. Chem. 265, 1360-1368 [Abstract/Free Full Text]
  21. Hon-nami, K., and Oshima, T. (1977) J. Biochem. (Tokyo) 82, 769-776 [Abstract]
  22. Xu, X., and Yagi, T. (1991) Biochem. Biophys. Res. Commun. 174, 667-672 [Medline] [Order article via Infotrieve]
  23. Papp, T., Kirchner, S., Diener, U., Jafari, M., Golka, A., and Schiffmann, D. (1995) Trends Genet. 11, 169 [CrossRef][Medline] [Order article via Infotrieve]
  24. Sambrook, J., Fritsch, F. E., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395 [Abstract]
  26. Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499 [Medline] [Order article via Infotrieve]
  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  28. Gornall, A. G., Bardawill, C. J., and David, M. M. (1949) J. Biol. Chem. 177, 751-766 [Free Full Text]
  29. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  30. Han, A.-L., Yagi, T., and Hatefi, Y. (1988) Arch. Biochem. Biophys. 267, 490-496 [Medline] [Order article via Infotrieve]
  31. Han, A.-L., Yagi, T., and Hatefi, Y. (1989) Arch. Biochem. Biophys. 275, 166-173 [Medline] [Order article via Infotrieve]
  32. Hekman, C., Tomich, J. M., and Hatefi, Y. (1991) J. Biol. Chem. 266, 13564-13571 [Abstract/Free Full Text]
  33. Doeg, K. A., and Ziegler, D. M. (1962) Arch. Biochem. Biophys. 97, 37-40 [Medline] [Order article via Infotrieve]
  34. Fogo, J. K., and Popowski, M. (1949) Anal. Biochem. 21, 732-737
  35. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  36. Kagawa, Y., Nojima, H., Nukiwa, N., Ishizuka, M., Nakajima, T., Yasuhara, T., Tanaka, T., and Oshima, T. (1984) J. Biol. Chem. 259, 2956-2960 [Abstract/Free Full Text]
  37. Shine, J., and Dalgarno, L. (1975) Nature 254, 34-38 [Medline] [Order article via Infotrieve]
  38. Faraldo, M. M., De Pedro, M. A., and Berenguer, J. (1992) J. Bacteriol. 174, 7458-7462 [Abstract]
  39. Ohnishi, T., Ragan, C. I., and Hatefi, Y. (1985) J. Biol. Chem. 260, 2782-2788 [Abstract]
  40. Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1996) J. Biol. Chem. 271, 5907-5913 [Abstract/Free Full Text]
  41. Deng, P. S., Hatefi, Y., and Chen, S. (1990) Biochemistry 29, 1094-1098 [Medline] [Order article via Infotrieve]
  42. Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) FEBS Lett. 354, 160-164 [CrossRef][Medline] [Order article via Infotrieve]
  43. Runswick, M. J., Gennis, R. B., Fearnley, I. M., and Walker, J. E. (1989) Biochemistry 28, 9452-9459 [Medline] [Order article via Infotrieve]
  44. Yano, T., Yagi, T., Sled', V. D., and Ohnishi, T. (1995) J. Biol. Chem. 270, 18264-18270 [Abstract/Free Full Text]
  45. Tran-Betcke, A., Warnecke, U., Bocker, C., Zaborosch, C., and Friedrich, B. (1990) J. Bacteriol. 172, 2920-2929 [Medline] [Order article via Infotrieve]
  46. Sled', V. D., Friedrich, T., Leif, H., Weiss, H., Meinhardt, S. W., Fukumori, Y., Calhoun, M. W., Gennis, R. B., and Ohnishi, T. (1993) J. Bioenerg. Biomembr. 25, 347-356 [Medline] [Order article via Infotrieve]
  47. Takano, S., Yano, T., and Yagi, T. (1996) Biochemistry 35, 9120-9127 [CrossRef][Medline] [Order article via Infotrieve]
  48. Masui, R., Wakabayashi, S., Matsubara, H., and Hatefi, Y. (1991) J. Biochem. (Tokyo) 110, 575-582 [Abstract]
  49. Albracht, S. P. J. (1993) Biochim. Biophys. Acta 1144, 221-224 [Medline] [Order article via Infotrieve]
  50. Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1995) Nature 373, 580-587 [CrossRef][Medline] [Order article via Infotrieve]
  51. Matsubara, H., and Saeki, K. (1992) Adv. Inorg. Chem. 38, 223-280
  52. Johnson, M. K. (1994) in Encyclopedia of Inorganic Chemistry (King, R. B., ed), Vol. 4, pp. 1896-1915, Wiley, Chichester, United Kingdom
  53. Yagi, T. (1991) J. Bioenerg. Biomembr. 23, 211-225 [Medline] [Order article via Infotrieve]
  54. Finel, M., Skehel, J. M., Albracht, S. P. J., Fearnley, I. M., and Walker, J. E. (1992) Biochemistry 31, 11425-11434 [Medline] [Order article via Infotrieve]
  55. Crouse, B. R., Yano, T., Finnegan, M. G., Yagi, T., and Johnson, M. K. (1994) J. Biol. Chem. 269, 21030-21036 [Abstract/Free Full Text]
  56. Golinelli, M.-P., Akin, L. A., Crouse, B. R., Johnson, M. K., and Meyer, J. (1996) Biochemistry 35, 8995-9002 [CrossRef][Medline] [Order article via Infotrieve]
  57. Conover, R. C., Kowal, A. T., Fu, W., Park, J.-B., Aono, S., Adams, M. W. W., and Johnson, M. K. (1990) J. Biol. Chem. 265, 8533-8541 [Abstract/Free Full Text]
  58. Collins, M. D., Shah, H. N., and Minnikin, D. E. (1980) J. Appl. Bacteriol. 48, 277-282 [Medline] [Order article via Infotrieve]
  59. Argos, P., Rossman, M. G., Frau, U. M., Zuber, H., Frank, G., and Tratschin, J. D. (1979) Biochemistry 18, 5698-5703 [Medline] [Order article via Infotrieve]
  60. Walker, J. E., Wonacott, A. J., and Harris, J. I. (1980) Eur. J. Biochem. 108, 581-586 [Abstract]
  61. Musafia, B., Buchner, V., and Arad, D. (1995) J. Mol. Biol. 254, 761-770 [CrossRef][Medline] [Order article via Infotrieve]
  62. Henning, M., Darimont, B., Sterner, R., Kirschner, K., and Jansonius, J. N. (1995) Structure 3, 1295-1306 [Abstract]
  63. Korndörfer, I., Steipe, B., Huber, R., Tomschy, A., and Jaenicke, R. (1995) J. Mol. Biol. 246, 511-521 [CrossRef][Medline] [Order article via Infotrieve]
  64. Yip, K. S. P., Stillman, T. J., Britton, K. L., Artymiuk, P. J., Baker, P. J., Sedelnikova, S. E., Engel, P. C., Pasquo, A., Chiaraluce, R., Consalvi, V., Scandurra, R., and Rice, D. W. (1995) Structure 3, 1147-1158 [Medline] [Order article via Infotrieve]
  65. Yoshida, M., Okamoto, H., Sone, N., Hirata, H., and Kagawa, Y. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 936-940 [Abstract]
  66. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.