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*

Takahiro YanoDagger §, Joseph SklarDagger , Eiko Nakamaru-Ogiso, Yasuhiro Takahashi||, Takao Yagi, and Tomoko OhnishiDagger

From the Dagger  Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the  Division of Biochemistry, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, and the || Department of Biology, Graduate School of Science, Osaka University, Osaka 560-0043, Japan

Received for publication, December 3, 2002, and in revised form, February 20, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 1×[2Fe-2S] and 2×[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 ~ 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 His106 with Cys specifically affected the fast-relaxing [4Fe-4S]1+ cluster, suggesting that this cluster is coordinated by His106. 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
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The energy-transducing NADH-quinone oxidoreductase is located at an entry point of the respiratory chain of mitochondria and bacteria, and catalyzes the redox coupled H+ or Na+ ion transport reaction (1). The enzyme complex is one of the largest and most complicated membrane-bound respiratory chain enzymes ever known. Mitochondrial enzyme (complex I) is composed of at least 46 subunits (2, 3), whereas the bacterial counterpart (NDH-1) such as those from Paracoccus denitrificans and Thermus thermophilus consists of only 14 subunits (4). All 14 subunits of the NDH-1 are homologous to their mitochondrial counterparts. Complex I/NDH-1 share many structural and enzymatic properties. Complex I/NDH-1 is composed of two distinct domains, a hydrophilic extramembrane domain (promontory part) and a hydrophobic membrane domain. Each domain plays a distinct role in the energy transducing reaction (5-9). Generally, the enzyme complexes contain the same type and number of redox components: one non-covalently bound FMN and up to eight iron-sulfur clusters (10). All of these redox cofactors are located in the promontory part where the oxidation of NADH and the subsequent electron transfer reaction takes place toward the membrane part. In the membrane part, a lipid-soluble quinone molecule accepts electrons. In this terminal reaction step, some membrane-bound quinone species are suggested to be involved (11, 12); however, these quinone-binding sites remain to be located. These redox reactions are endergonic, releasing free energy that is used to transport ions (H+ or Na+) through channels, which are thought to traverse the membrane domain. In the case of bovine heart mitochondrial complex I, ~4 H+ are transported during the electron transfer from NADH to quinone pool. However, the energy coupling mechanism has not been solved experimentally. The understanding of this process is a crucial issue of complex I bioenergetics.

Among the eight iron-sulfur clusters common in complex I/NDH-1 family, six clusters are generally detectable by EPR spectroscopy in situ, namely two [2Fe-2S] clusters, N1a and N1b, and four [4Fe-4S] clusters, N2, N3, N4, and N5 (10). Thus far, subunit locations of clusters N1a, N1b, N3, and N4 have tentatively been assigned based upon biochemical/physicochemical investigations of mammalian and microorganisms (10) and a series of expression studies of the individual putative iron-sulfur cluster-binding subunits of the P. denitrificans enzyme (13-15). Furthermore, two additional [4Fe-4S] clusters, which have not been detected by EPR in situ, are coordinated in the TYKY/Nqo9/NuoI subunit (nomenclature used for bovine heart mitochondria/P. denitrificans/Escherichia coli enzymes) with a primordial 2×[4Fe-4S] cluster-binding motif (16). These two [4Fe-4S] clusters have been investigated using optical/EPR spectroscopic techniques, revealing pH-independent thermodynamic properties in contrast to those expected for cluster N2 (17). These clusters were, therefore, tentatively designated N6a and N6b (17). A remaining putative iron-sulfur cluster binding subunit, PSST/Nqo6/NuoB subunit, shows significant sequence homology to a small subunit of [NiFe] hydrogenase family. It has been suggested that the PSST/Nqo6/NuoB subunit has played an evolutionarily central role along with 49-kDa/Nqo4/NuoD, TYKY/Nqo9/NuoI, ND1/Nqo8/NuoH, and ND5/Nqo12/NuoL subunits (18-20). Based on the sequence analysis and evolutionary relationship derived therefrom, it appears that the PSST/Nqo6/NuoB subunit accommodates a [4Fe-4S] cluster with a novel binding motif, which is suggested to be cluster N2 (21, 22). However, its final assignment remains to be experimentally made in the future.

The 75-kDa/Nqo3/NuoG subunit is the largest subunit and harbors three iron-sulfur cluster-binding motifs in the N-terminal region, containing 11 fully conserved cysteine (Cys37, Cys48, Cys51, Cys66, Cys110, Cys113, Cys119, Cys158, Cys161, Cys164, and Cys208; P. denitrificans numbering) and 1 fully conserved histidine residues (His106) (23-25). In the previous study, the Nqo3 subunit of P. denitrificans NDH-1 was expressed in E. coli, and the purified subunit was characterized in terms of iron-sulfur clusters (14). At least two iron-sulfur clusters, one [2Fe-2S] cluster (N1b) and one [4Fe-4S] cluster (N4), were detected (14). Although binding of a third iron-sulfur cluster in this subunit was suggested, clear evidence has not been presented until now. This postulated iron-sulfur cluster is thought to be a [4Fe-4S] cluster, N5, which has been detected in a limited number of complex I/NDH-1 family members. This elusive nature has cast a question on its identity as an intrinsic component of complex I/NDH-1. The N-terminal iron-sulfur cluster-binding domain of the Nqo3 subunit is highly homologous to some iron-sulfur cluster-binding proteins such as iron-only hydrogenase from Clostridium pasteurianum (Fig. 1). A high resolution x-ray crystal structure of C. pasteurianum iron-only hydrogenase clearly depicted that the corresponding 12 amino acid residues ligate one [2Fe-2S] cluster and two [4Fe-4S] clusters. Therefore, considering the high sequence similarity, it is very likely that the Nqo3 subunit can coordinate three iron-sulfur clusters in a similar manner.


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Fig. 1.   Comparison of the primary sequences of the N-terminal region of P. denitrificans Nqo3 subunit with those of its homologue subunits and hydrogenases. Amino acid sequences were obtained from GenBankTM: P. denitrificans Nqo3 subunit (1-223 amino acid residues, GenBankTM accession no. M84572), bovine heart mitochondrial complex I 75-kDa subunit (1-218 amino acid residues, GenBankTM accession no. A33552), E. coli NuoG subunit (1-219 amino acid residues, GenBankTM accession no. AE000317), C. pasteurianum Fe-only hydrogenase I (1-210 amino acid residues, GenBankTM accession no. AAA23248), and Thermotoga maritima Fe-hydrogenase alpha -subunit (1-206 amino acid residues, GenBankTM accession no. AAC02686). The sequences were aligned using ClastalW (53). Potential ligand residues for the three iron-sulfur clusters are shown with white letters in black boxes. Identical, strongly homologous, and weakly homologous amino acid residues are marked by *, :, and ., respectively. Sequence identity and homology of this region between P. denitrificans Nqo3 subunit and C. pasteurianum hydrogenase I are 25 and 43%, respectively.

In the present study, we re-investigated the iron-sulfur clusters bound in the P. denitrificans Nqo3 subunit expressed in E. coli. We employed the co-expression system using the iron-sulfur cluster biosynthesis (isc) genes from E. coli. The expressed Nqo3 subunit was found to contain the third iron-sulfur cluster, in addition to the previously identified clusters N1b and N4. The third cluster is a [4Fe-4S] type exhibiting novel EPR properties. Mutagenesis study suggests that this newly identified [4Fe-4S] cluster is coordinated by a mixed ligand, one histidine and three cysteine residues. Based on these results and others, we will discuss the identity of this novel [4Fe-4S] cluster as the elusive iron-sulfur cluster N5.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Constructions of Expression Vectors-- An expression vector for wild type Nqo3 subunit, pET11a/PdNqo3(wild), has been described in a previous report (14). Expression vectors for the site-directed mutants (H106A and H106C) were constructed as described below. The His106 right-arrow Ala substitution was introduced using Bio-Rad site-directed mutagenesis kit according to the protocol from the manufacturer. Single-strand DNA was prepared from E. coli strain CJ236 harboring the pTZ18U/PdNqo3(NdeI/BamHI) plasmid with M13KO7 helper phage. An oligonucleotide containing the following sequence was used: 5'-CTC ATC AAC GCT CCC CTG GAC-3' (altered sequences are underlined). For the H106C mutant, the GeneEditorTM in vitro site-directed mutagenesis system kit (Promega) was employed with the pTZ18U/PdNqo3(NdeI/BamHI) plasmid as a double-strand DNA template. The following mutation oligonucleotide was used: 5'-TTC CTG CTC ATC AAC TGT CCC CTG GAC TGC-3' (altered sequences are underlined). Clones obtained were verified by DNA sequencing for both strands. The NdeI/BamHI fragments containing the entire nqo3 gene were retrieved from the individual clones and were ligated into a pET11a expression plasmid cleaved with NdeI/BamHI. The final constructs were designated pET11a/PdNqo3(H106A) and pET11a/PdNqo3(H106C), respectively.

Expression of P. denitrificans Nqo3 Subunits with isc Genes in E. coli-- E. coli strain C41(DE3) was transformed with a vector pRKISC, which contained the entire isc gene operon (26). The E. coli strain containing pRKISC was subsequently transformed with pET11a/PdNqo3(wild), pET11a/PdNqo3(H106A), or pET11a/PdNqo3(H106C), and the transformants were grown on a LB plate containing 100 µg/ml ampicillin and 15 µg/ml tetracycline at 37 °C overnight. A well isolated colony was picked up and inoculated into 5 ml of LB medium containing 100 µg/ml ampicillin and 15 µg/ml tetracycline, and they were grown at 37 °C to the stationary phase. These cells were used to inoculate 1 liter of TB medium containing 100 µg/ml ampicillin, 15 µg/ml tetracycline, and 100 µM ammonium Fe(III) citrate, which was grown at 37 °C with good agitation (at 200 rpm) until its A600 nm reached ~0.5. Culture temperature was then lowered to 28 °C, and the cells were shaken at 100 rpm for 1 h. Isopropyl-1-thio-beta -D-galactopyranoside was added to 0.4 mM, and expression was induced for 14-16 h at 28 °C. The cells were harvested by centrifugation at 6000 rpm for 10 min at 4 °C. The cell pellets were immediately frozen and kept at -80 °C until use.

Purification of the Expressed P. denitrificans Nqo3 Subunit-- Purification of the Nqo3(wild) was performed in a COY anaerobic chamber (O2 < 1 ppm). All buffers used were repeatedly degassed in vacuum and purged with oxygen-free argon gas. The degassed buffers were equilibrated in the anaerobic chamber overnight. The cells (~20 g) were suspended in 100 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 1.0 mM DTT,1 1.0 mM EDTA, and 1.0 mM PMSF (Buffer A). The cell suspension was quickly passed through a French pressure cell at 1000 p.s.i. The treated cell suspension was immediately degassed in vacuum and purged with argon. Cytoplasmic fractions were obtained by ultracentrifugation at 45,000 rpm for 1 h at 4 °C and were applied onto a DEAE-Toyopearl column (3 × 5 cm) equilibrated with Buffer A. The column was washed with 100 ml of Buffer A. Retained proteins were eluted with a linear gradient of NaCl (0-0.5 M) in Buffer A. The Nqo3 subunit was eluted as a single peak with dark brown color. The fractions were pooled and directly applied onto a Bio-Rad HTP column (1 × 5 cm) equilibrated with 20 mM Tris-HCl (pH 7.4) buffer containing 1.0 mM DTT, 1.0 mM EDTA, 1.0 mM PMSF, and 0.25 M NaCl (Buffer B). The column was washed with 50 ml of Buffer B, and a linear gradient of potassium phosphate (0-0.2 M) in Buffer B was applied to elute bound proteins. Typically, the Nqo3(wild) subunit was eluted as a single sharp peak at a phosphate concentration of ~0.1 M. The most concentrated fractions were collected and diluted at least 5 times in Buffer A. The diluted proteins were applied onto a DEAE-Toyopearl column (0.5 × 2 cm). The adsorbed proteins were immediately eluted with Buffer A containing 0.5 M NaCl. Concentrated dark brown fractions were pooled in a test tube and were then applied onto a Sephacryl S-200 gel filtration column (1 × 35 cm) equilibrated with 20 mM Tris-HCl (pH 7.4) buffer containing 1.0 mM DTT, 1.0 mM EDTA, 1.0 mM PMSF, and 0.15 M NaCl (Buffer C). A brown peak was collected and concentrated with a small DEAE-Toyopearl column described above. The purified Nqo3(wild) subunit was further concentrated with a Microspin-30 to desired concentrations. The purified subunit (70-85% purity as judged by SDS-PAGE) was immediately used for characterizations. Purification of the Nqo3(H106C) variant was also carried out with the same protocol. Purification of Nqo3(H106A) was not possible because of poor incorporation of the iron-sulfur clusters, as is described under "Results."

Preparations of the Cholate-treated, NDH-1-enriched Cytoplasmic Membranes from P. denitrificans-- P. denitrificans was aerobically grown at 37 °C in synthetic medium with glucose as a carbon and energy source. The cytoplasmic membranes were prepared according to Ref. 27. The cytoplasmic membranes were further treated with potassium cholate in the presence of salts as described in Ref. 28. The membranes were suspended in 50 mM HEPES buffer (pH 7.7) containing 1.0 mM EDTA to 24 mg/ml and used for redox titration experiments.

Preparation of EPR Samples-- EPR samples were prepared as follows. The purified Nqo3 subunit (10-20 mg/ml) was placed in a microtube in an anaerobic chamber. Methyl viologen, benzyl viologen, and 4,4'-diethyl-1,1'-dimethylene-2,2'-dibromide were added to 100 µM each. Anaerobically prepared neutralized sodium dithionite solution (0.5 M) was added to reach 10 or 20 mM at a final concentration. The protein solution was transferred to an EPR tube, and after 5 min of incubation at room temperature the sample was quickly frozen in dry ice/alcohol mixture prior to liquid nitrogen before storage. For experiments to study pH effects, the highly concentrated Nqo3(wild) subunit (~30 mg/ml) was diluted 5 times in buffers containing 50 mM each of HEPES, MOPS, and Tris with pH adjusted to desired values with NaOH or HCl. To study effects of anti-freezing agents on EPR spectra, the Nqo3(wild) subunit (~30 mg/ml) was mixed with an equal volume of 100% ethylene glycol, 100% glycerol, or 1 M urea before reducing and freezing as described above.

Potentiometric redox titration of iron-sulfur clusters was carried out according to Ref. 29. The redox mediators used were 2-hydroxy-1,4-naphtoquinone, Indigo-2-sulfonate, Indigo-3-sulfonate, Indigo-4-sulfonate, safranin T, phenosafranin, methylviologen, benzyl viologen, triquat, 1,1-trimethylene-2,2'-dipyridinium dibromide, 4,4'-diethyl-1,1'-dimethylene-2.2'-dipyridium dibromide, neutral red, anthraquinone-2,6'-2-sulfonate, anthraquinone-2-sulfonate, and pycocyanin. Redox potentials (Eh) of the protein solutions were poised reductively with 0.5 or 0.1 M neutralized sodium dithionite solution and oxidatively with 0.1 M ferricyanide solution. Samples were anaerobically transferred into EPR tubes and were immediately frozen with dry ice/alcohol mix and then stored in liquid nitrogen until EPR analysis.

EPR Measurements-- EPR spectra were recorded by a Bruker ESP 300E spectrometer at X band (9.4 GHz) using an Oxford Instrument ESR900 helium flow cryostat, a Hewlett Packard 5350B microwave frequency counter, and an ITC4 temperature controller to control sample temperatures. EPR spectra of the iron-sulfur clusters were simulated by SimFonia (Bruker, Germany). Spin quantitations were carried out using 0.5 mM Cu(II)EDTA or 0.5 mM Cu(II) perchlorate as standards according to Ref. 30. Power saturation data were analyzed by a computer fitting method as described previously (31, 32).

Other Analytical Procedures-- Protein concentration was estimated by the method of Lowry et al. (33) in the presence of 1 mg/ml potassium-deoxycholate (34). Non-heme iron (35) and acid-labile sulfide contents (36, 37) were determined according to references cited. Any variations from the procedures and other details are described in the figure legends.

Materials-- Expression vector pET11a was purchased from Novagen. The E. coli strain C41(DE3) was a kind gift from Dr. John E. Walker (Medical Research Council, United Kingdom). All chemicals used were of the highest grade available from Sigma.

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Co-expression of the Nqo3 Subunit with the isc Genes in E. coli and Anaerobic Purification of the Subunit-- In the previous investigation, it had been shown that the expressed P. denitrificans Nqo3(wild) subunit contains one [2Fe-2S] cluster (g||,perp  = 2.03, 1.94) and one [4Fe-4S] cluster (gz,y,x = 2.06, 1.94, 1.88) (14). Based on spectral line shapes, these iron-sulfur clusters have tentatively been assigned to clusters N1b and N4, respectively. Although it is anticipated that the Nqo3 subunit could contain an additional iron-sulfur cluster, the previous study could not explicitly demonstrate the presence of a third iron-sulfur cluster. We reasoned that incorporation of iron-sulfur clusters into the expressed proteins might be limited by the overexpression system with E. coli used in the previous study. Furthermore, aerobic purification most likely resulted in oxidative destruction of the iron-sulfur clusters. The Nqo3 subunit is expressed in an apparently properly folded state. Therefore, if iron-sulfur cluster incorporation is improved, the subunit might be fully loaded with iron-sulfur clusters. Furthermore, anaerobic purification should prevent the oxidative damage of the iron-sulfur clusters even though all three iron-sulfur clusters might have been incorporated into the Nqo3 subunit in the previous study. In the present study, we have decided to employ a unique iron-sulfur cluster expression system, which has been successfully developed and utilized for the expression of several recombinant iron-sulfur proteins (26). In this system, a target iron-sulfur protein is expressed in E. coli with iron-sulfur cluster biosynthesis (isc) genes from E. coli. Amplification of the isc gene products supports biosynthesis and incorporation of iron-sulfur clusters into overexpressed proteins. When the P. denitrificans Nqo3(wild) subunit was expressed in E. coli strain C41(DE3) containing pRKISC, neither the expression level nor localization of the subunit was affected (data not shown). The Nqo3(wild) subunit was expressed as a water-soluble form in the cytoplasm and accounted for ~10% of the total cytoplasmic proteins estimated from SDS-PAGE analysis (data not shown). Significantly high incorporation of iron-sulfur cluster was evident from the fact that the prepared soluble fraction exhibited a very dark brown color. We carried out the purification of the expressed Nqo3(wild) subunit by conventional chromatography in a strictly anaerobic condition (see "Experimental Procedures"). The expressed Nqo3(wild) subunit was stable throughout purification, which was partly carried out at room temperature. The purified subunit with ligated iron-sulfur clusters stayed intact for a few weeks at room temperature in an anaerobic chamber.

EPR Characterization of the Iron-Sulfur Clusters in the Expressed P. denitrificans Nqo3 Subunit-- Non-heme iron and acid labile sulfide contents of the purified subunit were analyzed. The wild type Nqo3(wild) subunit yielded 6.8 ± 0.69 mol for both Fe and S2-/mol of subunit (an average of three independent preparations). These contents are remarkably improved from the previous experiments (14). To investigate whether the new preparation of the Nqo3 subunit contained the third iron-sulfur cluster, we reduced the purified subunit with 10 mM dithionite in the presence of redox mediators and thoroughly characterized the sample by EPR spectroscopy. At high temperatures near 40-60 K, EPR signals with axial symmetry (g||,perp  = 2.028, 1.940) were detected (Fig. 2A), arising from an S = 1/2 ground state of a [2Fe-2S]1+ cluster (N1b). The EPR line shape and spin relaxation behavior (P1/2 = 117 mW at 40 K) of this cluster are almost identical to those detected in the previous study. At 12 K, an EPR signal derived from an S = 1/2 ground state of a [4Fe-4S]1+ cluster was detected, which showed rhombic symmetry (gz,y,x = 2.068, 1.930, 1.895) (Fig. 2B). The half-saturation parameters (P1/2) of this cluster were determined as follows: P1/2 >200 mW at 20 K, 126 mW at 12 K, and 3-4 mW at 10 K. Below 10 K, the EPR signal was readily saturated. These properties are very similar to those of cluster N4 characterized in the previous study (14). These results indicate that the Nqo3(wild) subunit expressed in the new expression system also contains a [2Fe-2S] cluster (N1b) and a [4Fe-4S] cluster (N4).


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Fig. 2.   EPR spectra of the three iron-sulfur clusters detected in the dithionite-reduced P. denitrificans Nqo3(wild) subunit. Solid lines are experimental EPR data, and dashed lines are simulated spectra using EPR parameters described below. A, EPR spectrum of the [2Fe-2S]1+ cluster (N1b) in the S = 1/2 ground state detected at 40 K and 5 mW and the simulated spectrum with the parameters gz,y,x = 2.028, 1.940, 1.940, Lz,y,x = 24, 24, 24 gauss. B, EPR spectrum of the [4Fe-4S]1+ cluster (N4) in the S = 1/2 ground state detected at 12 K and 20 mW and the simulated spectrum with the parameters gz,y,x = 2.068, 1.930, 1.895, Lz,y,x = 19, 31, 33 gauss. C, EPR spectrum of the [4Fe-4S]fast1+ cluster in the S = 1/2 ground state detected at 5 K and 20 mW and the simulated spectrum with the parameters gz,y,x = 2.066, 1.915, 1.870, Lz,y,x = 33, 65, 70 gauss. EPR conditions used were as follows: microwave frequency, 9.44 GHz; modulation amplitude, 10 gauss; modulation frequency, 100 kHz; conversion time, 163 ms; and time constant, 163 ms. It should be noted that the simulated spectra shown here do not perfectly fit the experimental spectra using the method employed in this study (see "Experimental Procedures"). However, they are satisfactory enough to characterize the iron-sulfur clusters described here.

As the sample temperature was further lowered below 6 K, another EPR signal arising from an S = 1/2 [4Fe-4S]1+ cluster appeared (Fig. 2C). The EPR spectrum of this cluster exhibited broad and nearly axial symmetry with apparent g values of gz,y,x = 2.066, 1.915, 1.870 and with very broad line width (Lz,y,x = 33, 65, 70 gauss). This species showed a very fast spin relaxation at low temperature, P1/2 = 60 mW at 5 K (referred to as [4Fe-4S]fast hereafter). Midpoint redox potentials of all iron-sulfur clusters determined at pH 8.0 are very low. The [2Fe-2S] cluster (N1b) shows the lowest midpoint potential value, Em,8 = -472 mV, and the two [4Fe-4S] clusters have the almost same midpoint potentials, Em,8 = -363 mV for cluster N4 and Em,8 = -376 mV for the [4Fe-4S]fast cluster. We noticed that the [4Fe-4S]fast cluster is relatively unstable in fully reduced conditions. At very low potentials (Eh = ~-600 mV), the EPR signal decreased. This fragile nature is not observed either for the clusters N4 and N1b, both of which are stable under such conditions (data not shown). We estimated spin concentrations of the individual species observed in g = 2 region (S = 1/2 component only) in the fully reduced subunit by double integration of the simulated spectra of the individual species using Cu(II)EDTA or Cu(II) perchlorate as standards. The three iron-sulfur clusters were estimated to be present at a ratio of [2Fe-2S] (N1b):[4Fe-4S] (N4):[4Fe-4S]fast = 0.71:1.0:0.86.2 These biochemical and physicochemical analyses clearly demonstrate that the Nqo3(wild) subunit contains three dissimilar iron-sulfur clusters in the subunit.

Mixed Spin State (S = 1/2 + 3/2) of the [4Fe-4S]fast Cluster-- During the characterization of the preparations by EPR spectroscopy, we recognized that the Nqo3(wild) subunit exhibited an absorption-type EPR signal in a low magnetic field, g = 5 region (Fig. 3). The g = 5 signal was observed only at a low temperature range, 4-6 K, and was more distinct at microwave power levels higher than 50 mW. The g = 5 signal is characteristic of the S = 3/2 spin system of Kramer's doublet. The other two g components could not be identified because either those effective g values are too low or such signals were overlapped with other EPR signals. The S = 3/2 EPR signal most likely arises from the [4Fe-4S]fast cluster because the signal was not detected in previous preparations that harbored only cluster N1b and cluster N4 (data not shown). The signal line shape is rather broad, such that we could not resolve any structural feature in temperature-dependent experiments to obtain information on the zero-field splitting ground state. It should be noted that appearance of this signal differs from one preparation to the other. In many samples we examined, the S = 1/2 component was a dominant species. We examined effects of the addition of anti-freezing reagents such as glycerol and ethylene glycol on EPR signals, which are known to affect the spin state of nitrogenase iron-protein of Azotobacter vinelandii (38). Although these reagents affected EPR line shape of clusters N1b and N4, particularly on their line widths (data not shown), we were not able to see any specific changes in the spin-state of either [4Fe-4S]fast cluster or other clusters. Currently it is still not clear as to what affects the spin state of this [4Fe-4S]fast cluster. It appears that the spin state of the cluster is sensitive to environments and/or to subtle changes in protein conformation. To clarify factors influencing the spin states of the cluster, further experiments are currently under way. The g = 4.3 signal observed in the same magnetic field seems to be derived from Fe(III) ions non-specifically bound to or contaminated in the Nqo3 subunit preparations.


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Fig. 3.   EPR spectrum of the dithionite-reduced P. denitrificans Nqo3(wild type) subunit in a lower magnetic field (g ~ 5 region). The spectrum was measured at 5 K and microwave power level of 100 mW. EPR spectrum in a wide magnetic field was shown in left inset. A high spin EPR signal at g = 4.3 seems to arise from Fe(III) ions in a rhombic symmetry associated with the proteins or as contaminants in the sample. The simulated spectrum of the Fe(III) ions was shown by broken line. The parameters used are gz,y,x = 4.363, 4.236, 4.236, Lz,y,x = 12, 105, 75 gauss. Right inset shows a difference spectrum obtained by subtracting the simulated spectrum of Fe(III) ions from the experimental spectrum. EPR conditions used were the same as in Fig. 2.

Site-directed Mutation of the Fully Conserved His106 of the Nqo3 Subunit-- To examine whether the fully conserved histidine residue is an actual ligand of one of three iron-sulfur clusters, we mutated His106 to Ala or Cys, and the mutated subunits were expressed in E. coli. When His106 was replaced by an alanine residue, the Nqo3(H106A) subunit expression was normal in the cytoplasmic fraction (data not shown) but the incorporation of iron-sulfur clusters was significantly decreased. Partially purified subunits were very faintly brown or almost completely colorless. Even bound iron-sulfur clusters were so unstable that they were readily lost during the first chromatography, even though the preparations were maintained in strictly anaerobic conditions. Therefore, we could not obtain preparations that could be further studied. These observations suggest that the His106 residue plays an important role in coordinating the iron-sulfur clusters.

When the histidine was mutated to cysteine residue, the expressed subunit retained all three iron-sulfur clusters. The Nqo3(H106C) subunit contained both clusters N1b and N4. The signal line shape and relaxation properties of the cluster N1b were virtually identical to those in the wild type subunit (Fig. 4A; see also Fig. 2A). Although slight broadening of its spectral line shape and shift of the signal peak positions were noticeable, the mutated subunit exhibited EPR signals comparable with those arising from cluster N4 in the wild type subunit (Fig. 4B). However, the [4Fe-4S]fast cluster was significantly affected by this substitution. Heterogeneity was readily recognized; at 5 K and 10 mW, the gx signal was significantly broadened (Fig. 4C). At least two different subspecies with gz = 2.075 and gz = 2.057 were detected at 20 mW of incident microwave power level at 5 K (Fig. 4C, inset). It was also observed that all iron-sulfur clusters coordinated in this mutated subunit were very unstable. The iron-sulfur clusters were lost in a few days even in an anaerobic condition in contrast to the wild type Nqo3 subunit. These results clearly demonstrate that the substitution of His106 to Cys specifically affects the [4Fe-4S]fast cluster, and interestingly such alterations substantially influence the stability of two other iron-sulfur clusters. Taken together, it is conceivable that the His106 coordinates the [4Fe-4S]fast cluster.


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Fig. 4.   EPR spectra of the three iron-sulfur clusters detected in the dithionite-reduced P. denitrificans Nqo3(H106C) mutant subunit. A, EPR spectrum of the S = 1/2 [2Fe-2S]1+ cluster (N1b) detected at 40 K and 5 mW. B, EPR spectrum of the S = 1/2 [4Fe-4S]1+ cluster (N4) detected at 12 K and 20 mW. C, EPR spectrum of the S = 1/2 [4Fe-4S]fast1+ cluster detected at 5 K and 10 mW. Significant heterogeneities were seen at gz and gx components (indicated by open arrows). EPR signals around gz components of the [4Fe-4S] species are shown in inset. The spectrum was measured at 5 K and 20 mW. All other EPR conditions were the same as in Fig. 2.

EPR Characterization of Iron-Sulfur Clusters in the Cholate-treated, NDH-1-enriched P. denitrificans Membranes in Situ-- Previously, EPR signals derived from clusters N1a, N1b, N2, N3, and N4 were identified in the cytoplasmic membrane of P. denitrificans (39). Because purification of an intact NDH-1 enzyme complex from P. denitrificans has not been successful thus far, detailed analysis of the bound iron-sulfur clusters in the isolated NDH-1 has not been conducted. As reported earlier, the NDH-1 content in the P. denitrificans cytoplasmic membrane can be increased up to 3-5 times higher by treating the membranes with potassium cholate in the presence of salt (28). This treatment does not seem to alter structural and enzymatic properties of the in situ NDH-1 significantly. Taking advantage of the NDH-1-enriched membrane preparation, we re-characterized iron-sulfur clusters associated with NDH-1 by EPR spectroscopy. As expected, we could observe higher intensity EPR signals arising from iron-sulfur clusters in the NDH-1 in cholate-treated membranes. We detected EPR signals from most of the iron-sulfur clusters previously identified, namely those of clusters N1b, N2, N3, and N4 (39). EPR properties and midpoint redox potentials are summarized in Table I (also see Fig. 5). Cluster N1b showed EPR signal with axial symmetry, and its Em value was determined to be Em,7.7 = -270 mV. In contrast to the previous study, we were unable to identify the EPR signal arising from cluster N1a within the Eh range between -418 mV and -1 mV. The Em,7.0 value was previously reported to be -150 mV in membrane preparations, which is higher than those of other complex I/NDH-1 species. Recently, the redox properties of cluster N1a in the expressed Nqo2 subunits (from P. denitrificans and T. thermophilus) and in the expressed 24-kDa subunit (from bovine heart mitochondrial complex I) have been studied using cyclic voltammetry (40). The Em values of these expressed subunits are all low, approximately -400 mV. It has been revealed that thermodynamic properties of cluster N1a are sensitive to environments such as pH and salt concentrations. Therefore, the redox potential of cluster N1a might have significantly dropped in the preparation studied in the present investigation. To address this issue, more careful examinations are currently under way in our laboratories. Clusters N3 and N4 exhibit EPR spectra with apparent g values of gz,y,x = 2.03, 1.94, 1.87 and gz,y,x = 2.10, 1.94, 1.88, respectively (Fig. 5). The gz value of cluster N3 was reported to be gz = 2.01 in the previous study using a cytoplasmic membrane preparation without the NDH-1 enrichment treatment (39). The midpoint redox potentials were determined to be Em,7.7 = -285 mV for cluster N3 and Em,7.7 = -310 mV for cluster N4, which are almost identical to those of the previous report within experimental error. Cluster N2 shows an axial EPR signal with g||,perp  = 2.055, 1.92, and its midpoint redox potential was estimated to be Em,7.7 = -160 mV. Furthermore, we recognized additional EPR signals arising from a [4Fe-4S] cluster in this preparation. As shown in Fig. 5, this species becomes visible at lower temperatures and exhibits a rather broad gz signal at g = 2.059. In addition, a signal was also noticeable at gx = 1.865 at 4 K. Because of the overlap with other iron-sulfur cluster signals, it was not possible to resolve its gy component (gy = ~1.94). The midpoint redox titration of this [4Fe-4S] cluster was estimated to be Em,7.7 = -250 mV. The EPR and thermodynamic properties presented above are very similar to those of cluster N5, which have been reported for mitochondrial complex I from bovine heart (41) and Yarrowia lipolytica (9). It should be noted that a spin concentration of this [4Fe-4S] cluster detected in the P. denitrificans membranes appears to be stoichiometrically not equivalent to those of other iron-sulfur clusters associated with NDH-1 such as clusters N3 and N4, which is similar to the case of Y. lipolytica complex I (42). We should also point out that the overall EPR properties of this newly identified [4Fe-4S] cluster in the P. denitrificans membrane in situ are similar to those of the [4Fe-4S]fast cluster in the expressed subunit described above. Based on these substantial similarities, we propose that the [4Fe-4S] cluster housed in the Nqo3 subunit corresponds to cluster N5 in the NDH-1 enzyme complex.


                              
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Table I
Apparent g values and midpoint redox potentials at pH 7.7 of the iron-sulfur clusters of the P. denitrificans NDH-1 in membranes


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Fig. 5.   EPR spectra of the g = 2 region of the cholate-treated, NDH-1-enriched P. denitrificans membranes measured at different sample temperatures. A redox potential (Eh) of the membrane suspension was potentiometrically poised at Eh = -437 mV in the presence of redox mediators as described in detail under "Experimental Procedures." EPR spectra were measured at 10 mW. EPR signals arising from iron-sulfur clusters, N2, N3, and N4 are indicated by solid arrows with apparent g values. At 4 K, EPR signals previously unidentified were detected at g = 2.059 and 1.865 (shown by open arrows).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, iron-sulfur cluster incorporation into the expressed Nqo3 subunit was significantly improved by co-expression with E. coli iron-sulfur cluster biosynthesis (isc) genes. The Nqo3(wild) subunit harbored three iron-sulfur clusters with stoichiometric amounts, which allowed us to characterize the third iron-sulfur cluster coordinated in the subunit for the first time, in addition to the previously identified clusters N1b and N4 (14). Although the sequence analysis has predicted such a possibility, the third iron-sulfur cluster was not experimentally identified in the Nqo3/NuoG/75-kDa subunit before (14, 23, 43). The present study revealed that the third iron-sulfur cluster is a [4Fe-4S] type, showing unique EPR properties. EPR signals from this [4Fe-4S] cluster (referred to as [4Fe-4S]fast) are detectable only at extremely low temperatures. The EPR spectral line shape is relatively broad and nearly axial (with apparent g values of gz,y,x = 2.066, 1.915, 1.870, gav = ~1.95). Our EPR analysis also revealed a unique feature that an unpaired electron of the [4Fe-4S]fast cluster can exist in a mixed spin ground state (S = 1/2 + 3/2). These observations provided the experimental basis to discuss the elusive iron-sulfur cluster N5 of the complex I/NDH-1 (see below).

Mutagenensis experiments revealed that the mutation of the fully conserved His106 to Cys resulted in the specific and significant alteration of EPR properties of the [4Fe-4S]fast cluster, indicating that His106 most likely ligates to the [4Fe-4S]fast cluster. In this mutant, the substituted Cys, which is generally a more suitable ligand of iron-sulfur clusters, seemed to ligate to the iron-sulfur cluster. However, the H106C mutant could not maintain the stability of the iron-sulfur clusters as in the wild type subunit (44). The loss of ligand in the H106A mutant resulted in almost no incorporation of all iron-sulfur clusters into the subunit. These results clearly indicate that the ligation of [4Fe-4S]fast is crucial for the incorporation and stabilization of the neighboring iron-sulfur clusters in the Nqo3 subunit. It is interesting that His106 is a more suitable ligand than cysteine in this case. This suggests that the His plays a structurally and functionally important role because this His is evolutionarily conserved not only in the Nqo3 subunit homologues of complex I/NDH-1 but also in the hydrogenase family. In the x-ray crystal structure of the iron-only hydrogenase from C. pasteurianum, the corresponding iron-sulfur cluster binding domain harbors three iron-sulfur clusters, one binuclear (FS2) and two tetranuclear iron-sulfur clusters (FS4B and FS4C) (Fig. 6; see also Fig. 1) (45). The FS2, FS4B, and FS4C appear to correspond to clusters N1b, N4, and [4Fe-4S]fast in the Nqo3 subunit, respectively. The FS4C is coordinated by the amino acid residues His94, Cys98, Cys101, and Cys107 (C. pasteurianum numbering; see Fig. 1). These amino acid residues are located in a loop connecting two alpha -helices, and their side chains are pointing inward to ligate the [4Fe-4S] cluster. Interestingly, His94 is not only coordinating the cluster FS4C but also interacting with its neighboring sites through a hydrogen-bonding network. In the C. pasteurianum structure, the delta N of the His94 is hydrogen-bonded with backbone carbonyl oxygen of Leu148 through a water molecule. This Leu148 is adjacent to the Cys147 ligand of the FS4B. This hydrogen bond network has been proposed to be a potential electron transfer pathway between the FS4B and FS4C clusters (45). Considering the high sequence similarity between iron-sulfur cluster binding domains of these two proteins, it seems very likely that the iron-sulfur cluster-binding domain of the Nqo3 subunit takes a similar structural coordination. Indeed, our data obtained in mutagenesis experiments appear to support this assumption and provide additional insight into the specific structural and functional role of the conserved histidine. The replacement of the His106 by Cys of the Nqo3 subunit still allows binding of the [4Fe-4S]fast cluster (Fig. 4C). This substitution results in the loss of the hydrogen bond interaction with its neighboring cluster N4. Although the H106C subunit exhibited EPR signals arising from cluster N4, the signals were noticeably perturbed (Fig. 4B). Conversely, the EPR spectrum of cluster N1b, which is far from the [4Fe-4S]fast cluster, is virtually identical to that of the wild type subunit. These observations can be taken as evidence that the loss of the hydrogen bond network affects the properties of cluster N4. This modified interaction between these two iron-sulfur cluster-binding domains seems to eventually lead to destabilization of all iron-sulfur clusters. It is reasonable to infer that the situation is not ameliorated in the H106A mutant. Inability to coordinate the [4Fe-4S]fast cluster can cause disastrous effects on the iron-sulfur cluster binding domain, leading to poor ligation of all the iron-sulfur clusters in the Nqo3 subunit in the expression system. In any case, the His ligation to the fast-relaxing [4Fe-4S] cluster deserves more detailed magnetic resonance study in the future.


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Fig. 6.   A close up of the iron-sulfur cluster binding domain of the C. pasteurianum Fe-only hydrogenase I three-dimensional x-ray crystallographic structure. Amino acid residues coordinating the iron-sulfur clusters, FS2, FS4B, and FS4C, are shown (also see Fig. 1). The figure was drawn by Rasmol using a coordinate for the C. pasteurianum iron-only hydrogenase (Protein Data Bank identification no. 1FEH).

It has been questioned whether cluster N5 is an actual intrinsic component of complex I/NDH-1. From the present study it is apparent that the Nqo3/NuoG/75-kDa subunit is able to house an additional [4Fe-4S] cluster. Based on the unique physicochemical properties discussed below, it is possible that the bound [4Fe-4S] cluster corresponds to cluster N5. Thus far, EPR signals of cluster N5 have been detected in complex I/NDH-1 from only a few organisms. Cluster N5 was first detected in pigeon heart mitochondria with Em = -260 mV, but at a substoichiometric amount (0.25 spin per cluster N2) (41). Later, an EPR signal derived from cluster N5 was detected in Rhodobacter sphaeroides NDH-1 (46); and, more recently, cluster N5 EPR signal was detected in the isolated complex I from Y. lipolytica mitochondria with a substoichiometric spin concentration (9, 42). In a well characterized mitochondrial complex I from Neurospora crassa, cluster N5 has not been detected (47). A property common among cluster N5 detected in these species is that the spin relaxation is significantly fast so that the EPR signals are detectable only at extremely low temperatures. In the present study, we detected EPR signals very homologous to those of cluster N5 in similar conditions in the P. denitrificans membranes in which the NDH-1 content had been significantly increased by mild detergent treatments. The midpoint potential of the cluster is Em = -250 mV, which is similar to that of cluster N5 in bovine heart mitochondrial complex I. The present study demonstrated that the [4Fe-4S]fast species in the expressed Nqo3 subunit also shares similar properties with cluster N5 detected in organisms mentioned above, strongly suggesting that cluster N5 is coordinated in the Nqo3/NuopG/75-kDa subunit.

The present study further revealed a novel property that the [4Fe-4S]fast cluster exhibits mixed spin ground states (S = 1/2 + 3/2). The iron-sulfur clusters of the structurally very similar C. pasteurianum iron-only hydrogenase have been extensively investigated by EPR and magnetic circular dichroism spectroscopy. The dithionite-reduced hydrogenase exhibited a broad featureless EPR resonance in the S = 1/2 region, which arose from magnetically interacting multiple iron-sulfur clusters, one [2Fe-2S] (FS2) and three [4Fe-4S] clusters (FS4A, FS4B, and FS4C) (48, 49). In addition, because the midpoint redox potentials of these clusters are very similar (Em,8 -400 mV) (48), it was not successful to resolve EPR spectra of the individual iron-sulfur clusters. The following low temperature magnetic circular dichroism and EPR study of the C. pasteurianum hydrogenase has indicated that one of three [4Fe-4S] clusters exhibited the S = 3/2 ground state (50). Although it is not known which [4Fe-4S] cluster exhibits such a unique spin state, it may emulate that the corresponding [4Fe-4S] cluster (FS4C) with a histidine ligand may be responsible for the existence of the higher spin ground state. In general, physiological significance of higher electronic spin states of iron-sulfur clusters is not understood yet. However, the finding of the presence of the S = 3/2 ground state [4Fe-4S] cluster in the expressed Nqo3 subunit can at least provide an empirical explanation on the elusive nature of cluster N5. Extremely fast-relaxing [4Fe-4S]1+ clusters are difficult to investigate with certainty. For a detection of spins in the higher spin ground state such as S = 3/2 state, significantly high protein concentrations are required, which is generally difficult to achieve, particularly for large transmembrane enzyme complexes like complex I/NDH-1. As observed in the present study, the spin state of the [4Fe-4S]fast cluster seems to be sensitive to subtle changes in protein conformation and/or environments. If some structural changes occur in the iron-sulfur cluster binding domain of the subunit, then it may affect the spin state of the [4Fe-4S]fast cluster, altering a ratio of the ground state population between S = 1/2 and S = 3/2 states. It should be noted that structural changes seem to take place around this subunit of the enzyme complex in situ. Studies on cross-linking and susceptibility to trypsin digestion of mitochondrial complex I have shown that extensive conformational changes undergo upon reduction by substrate NADH or NADPH (51, 52). These physicochemical features of the higher spin system and associated technical difficulties may partly explain why cluster N5 is only partially or hardly detectable in complex I/NDH-1 from different species.

    ACKNOWLEDGEMENTS

We thank Jack Catalano and Rishi Porecha for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01GM30736 (to T. O.) and R01GM33712 (to T. Y.).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.

§ To whom correspondence should be addressed. Tel.: 215-898-2939; Fax: 215-573-3748; E-mail: yano@mail.med.upenn.edu.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212275200

2 Spin concentrations of the individual iron-sulfur clusters were determined with the purified Nqo3(wild) subunit preparations in which the [4Fe-4S]fast1+ cluster dominantly exhibited EPR signals from S = 1/2 ground state. Accurate quantitation of the g = 5 signal arising from S = 3/2 ground state in the same sample was difficult because of the small signal amplitude.

    ABBREVIATIONS

The abbreviations used are: DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; W, watt(s); MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Yano, T. (2002) Mol. Aspects Med. 23, 345-368[CrossRef][Medline] [Order article via Infotrieve]
2. Walker, J. E. (1992) Q. Rev. Biophys. 25, 253-324[Medline] [Order article via Infotrieve]
3. Carroll, J., Shannon, R. J., Fearnley, I. M., Walker, J. E., and Hirst, J. (2002) J. Biol. Chem. 277, 50311-50317[Abstract/Free Full Text]
4. Yagi, T., Yano, T., Di Bernardo, S., and Matsuno-Yagi, A. (1998) Biochim. Biophys. Acta 1364, 125-133[Medline] [Order article via Infotrieve]
5. Guènebaut, V., Vincentelli, R., Mills, D., Weiss, H., and Leonard, K. R. (1997) J. Mol. Biol. 265, 409-418[CrossRef][Medline] [Order article via Infotrieve]
6. Guènebaut, V., Schlitt, A., Weiss, H., Leonard, K. R., and Friedrich, T. (1998) J. Mol. Biol. 276, 105-112[CrossRef][Medline] [Order article via Infotrieve]
7. Grigorieff, N. (1998) J. Mol. Biol. 277, 1033-1046[CrossRef][Medline] [Order article via Infotrieve]
8. Friedrich, T. (1998) Biochim. Biophys. Acta 1364, 134-146[Medline] [Order article via Infotrieve]
9. Djafarzadeh, R., Kerscher, S., Zwicker, K., Radermacher, M., Lindahl, M., Schagger, H., and Brandt, U. (2000) Biochim. Biophys. Acta 1459, 230-238[Medline] [Order article via Infotrieve]
10. Ohnishi, T. (1998) Biochim. Biophys. Acta 1364, 186-206[Medline] [Order article via Infotrieve]
11. Magnitsky, S., Toulokhonova, L., Yano, T., Sled, V. D., Hagerhall, C., Grivennikova, V. G., Burbaev, D. S., Vinogradov, A. D., and Ohnishi, T. (2002) J. Bioenerg. Biomembr. 34, 193-208[CrossRef][Medline] [Order article via Infotrieve]
12. Yano, T., Magnitsky, S., and Ohnishi, T. (2000) Biochim. Biophys. Acta 1459, 299-304[CrossRef][Medline] [Order article via Infotrieve]
13. Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499[Medline] [Order article via Infotrieve]
14. Yano, T., Yagi, T., Sled', V. D., and Ohnishi, T. (1995) J. Biol. Chem. 270, 18264-18270[Abstract/Free Full Text]
15. Yano, T., Sled, V. D., Ohnishi, T., and Yagi, T. (1996) J. Biol. Chem. 271, 5907-5913[Abstract/Free Full Text]
16. Yano, T., Magnitsky, S., Sled', V. D., Ohnishi, T., and Yagi, T. (1999) J. Biol. Chem. 274, 28598-28605[Abstract/Free Full Text]
17. Rasmussen, T., Scheide, D., Brors, B., Kintscher, L., Weiss, H., and Friedrich, T. (2001) Biochemistry 40, 6124-6131[CrossRef][Medline] [Order article via Infotrieve]
18. Friedrich, T., and Weiss, H. (1997) J. Theor. Biol. 187, 529-540[CrossRef][Medline] [Order article via Infotrieve]
19. Friedrich, T., and Scheide, D. (2000) FEBS Lett. 479, 1-5[CrossRef][Medline] [Order article via Infotrieve]
20. Ahlers, P. M., Zwicker, K., Kerscher, S., and Brandt, U. (2000) J. Biol. Chem. 275, 23577-23582[Abstract/Free Full Text]
21. Albracht, S. P. (1993) Biochim. Biophys. Acta 1144, 221-224[Medline] [Order article via Infotrieve]
22. Yano, T., and Ohnishi, T. (2001) J. Bioenerg. Biomembr. 33, 213-222[CrossRef][Medline] [Order article via Infotrieve]
23. Xu, X., Matsuno-Yagi, A., and Yagi, T. (1992) Arch. Biochem. Biophys. 296, 40-48[Medline] [Order article via Infotrieve]
24. Runswick, M. J., Gennis, R. B., Fearnley, I. M., and Walker, J. E. (1989) Biochemistry 28, 9452-9459[Medline] [Order article via Infotrieve]
25. Yano, T., Chu, S. S., Sled', V. D., Ohnishi, T., and Yagi, T. (1997) J. Biol. Chem. 272, 4201-4211[Abstract/Free Full Text]
26. Nakamura, M., Saeki, K., and Takahashi, Y. (1999) J. Biochem. (Tokyo) 126, 10-18[Abstract]
27. Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311[Medline] [Order article via Infotrieve]
28. Takano, S., Yano, T., and Yagi, T. (1996) Biochemistry 35, 9120-9127[CrossRef][Medline] [Order article via Infotrieve]
29. Dutton, P. L. (1978) Methods Enzymol. 54, 411-435[Medline] [Order article via Infotrieve]
30. Aasa, R., and Vänngård, T. (1975) J. Magn. Res. 19, 308-315
31. Vinogradov, A. D., Sled, V. D., Burbaev, D. S., Grivennikova, V. G., Moroz, I. A., and Ohnishi, T. (1995) FEBS Lett. 370, 83-87[CrossRef][Medline] [Order article via Infotrieve]
32. Rupp, H., Rao, K. K., Hall, D. O., and Cammack, R. (1978) Biochim. Biophys. Acta 537, 255-260[Medline] [Order article via Infotrieve]
33. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
34. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250[Medline] [Order article via Infotrieve]
35. Fish, W. W. (1988) Methods Enzymol. 158, 357-364[Medline] [Order article via Infotrieve]
36. Beinert, H. (1978) Methods Enzymol. 54, 435-445[Medline] [Order article via Infotrieve]
37. Fogo, J. K., and Popowski, M. (1949) Anal. Biochem. 21, 732-737
38. Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Munck, E. (1985) J. Biol. Chem. 260, 11160-11173[Abstract/Free Full Text]
39. Meinhardt, S. W., Kula, T., Yagi, T., Lillich, T., and Ohnishi, T. (1987) J. Biol. Chem. 262, 9147-9153[Abstract/Free Full Text]
40. Zu, Y., Di Bernardo, S., Yagi, T., and Hirst, J. (2002) Biochemistry 41, 10056-10069[CrossRef][Medline] [Order article via Infotrieve]
41. Ohnishi, T. (1975) Biochim. Biophys. Acta 387, 475-490[Medline] [Order article via Infotrieve]
42. Kerscher, S., Drose, S., Zwicker, K., Zickermann, V., and Brandt, U. (2003) Biochim. Biophys. Acta 155, 83-91
43. Pilkington, S. J., Skehel, J. M., Gennis, R. B., and Walker, J. E. (1991) Biochemistry 30, 2166-2175[Medline] [Order article via Infotrieve]
44. Gutierrez-Correa, J., Fairlamb, A. H., and Stoppani, A. O. (2001) Free Radic. Res. 34, 363-378[Medline] [Order article via Infotrieve]
45. Peters, J. W., Lanzilotta, W. N., Lemon, B. J., and Seefeldt, L. C. (1998) Science 282, 1853-1858[Abstract/Free Full Text]
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. Weiss, H., Friedrich, T., Hofhaus, G., and Preis, D. (1991) Eur. J. Biochem. 197, 563-576[Medline] [Order article via Infotrieve]
48. Adams, M. W. (1987) J. Biol. Chem. 262, 15054-15061[Abstract/Free Full Text]
49. Fu, W., Drozdzewski, P. M., Morgan, T. V., Mortenson, L. E., Juszczak, A., Adams, M. W., He, S. H., Peck, H. D., Jr., DerVartanian, D. V., LeGall, J., and Johnson, M. K. (1993) Biochemistry 32, 4813-4819[Medline] [Order article via Infotrieve]
50. Zambrano, I. C., Kowal, A. T., Mortenson, L. E., Adams, M. W., and Johnson, M. K. (1989) J. Biol. Chem. 264, 20974-20983[Abstract/Free Full Text]
51. Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576[Medline] [Order article via Infotrieve]
52. Yamaguchi, M., Belogrudov, G. I., and Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098[Abstract/Free Full Text]
53. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract]


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