©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression and Characterization of the 66-Kilodalton (NQO3) Iron-Sulfur Subunit of the Proton-translocating NADH-Quinone Oxidoreductase of Paracoccus denitrificans(*)

(Received for publication, April 3, 1995; and in revised form, May 4, 1995)

Takahiro Yano Takao Yagi Vladimir D. Sled' Tomoko Ohnishi

From the  (1)Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 (2)Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 dissimilar subunits which are designated NQO1-14 and contains one noncovalently bound FMN and at least five EPR-visible iron-sulfur clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of the deduced primary structures of the subunits with consensus sequences for the cofactor binding sites has predicted that NQO1, NQO2, NQO3, NQO9, and probably NQO6 subunits are cofactor binding subunits. Previously, we have reported that the NQO2 (25 kDa) subunit was overexpressed as a water-soluble protein in Escherichia coli and was found to ligate a single [2Fe-2S] cluster with rhombic symmetry (g = 1.92, 1.95, and 2.00) (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T.(1994) Biochemistry 33, 494-499). In the present study, the NQO3 (66 kDa) subunit, which is equivalent to the 75-kDa subunit of bovine heart Complex I, was overexpressed in E. coli. The expressed NQO3 subunit was found predominantly in the cytoplasmic phase and was purified by ammonium sulfate fractionation and anion-exchange chromatography. The chemical analyses and UV-visible and EPR spectroscopic studies showed that the expressed NQO3 subunit contains at least two distinct iron-sulfur clusters: a [2Fe-2S] cluster with axial EPR signals (g = 1.934 and 2.026, and L = 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster with rhombic symmetry (g = 1.892, 1.928, and 2.063, and L = 2.40, 1.55, and 1.75 millitesla). The midpoint redox potentials of [2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are -472 and -391 mV, respectively. The tetranuclear cluster in the isolated NQO3 subunit is sensitive toward oxidants and converts into [3Fe-4S] form. The assignment of these iron-sulfur clusters to those identified in the P. denitrificans NDH-1 enzyme complex and the possible functional role of the NQO3 subunit is discussed.


INTRODUCTION

It is generally accepted that there are two types of NADH-quinone (Q) (^1)oxidoreductase in bacterial respiratory chains (see Yagi(1993)). One is not an energy-coupling enzyme and is designated NDH-2. The NDH-2 enzyme contains only a noncovalently bound FAD as a cofactor, and is composed of a single polypeptide (Jaworowski et al., 1981). The enzymes capable of energy transformation can be further divided into two types. One type, designated as Na-NDH, has been found in marine alkalophilic microbes and is capable of translocating sodium ion as a coupling ion. It is composed of three different subunits and contains one FMN and one FAD as prosthetic groups (see Unemoto and Hayashi (1993)). The other type represents the multisubunit enzyme complex which can function as a proton pump and generates an electrochemical proton gradient across the plasma membrane, and is designated NDH-1. The NDH-1 contains a noncovalently bound FMN and several iron-sulfur clusters as cofactors and is composed of at least 14 dissimilar subunits (Yagi et al., 1992, 1993; Weidner et al., 1993; Leif et al., 1995). In terms of polypeptide composition, cofactors, and enzymatic properties, the bacterial NDH-1 enzyme complex appears to be the counterpart of the mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (Complex I).

The Paracoccus denitrificans NDH-1 is one of the well characterized enzyme complexes of bacterial NDH-1. It has been isolated from membrane and characterized (Yagi, 1986), and was shown to be similar to the mitochondrial Complex I in terms of enzymatic properties and immuno cross-reactivity. The structural genes encoding the P. denitrificans NDH-1 have been cloned and sequenced (Xu et al., 1991a, 1991b, 1992a, 1992b, 1993). It was found that these genes constitute a cluster composed of 14 structural genes and 6 unidentified reading frames (URFs), which are designated NQO1-14 and URF1-6, respectively (Yagi et al., 1992). All of the 14 subunits are highly homologous to their counterparts in bovine heart Complex I, which is composed of at least 41 subunits (Walker, 1992). In addition, the P. denitrificans NDH-1 shows striking EPR spectral similarity to the bovine heart Complex I. These facts make the P. denitrificans NDH-1 a useful model system for study of the structure and mechanism of action of mitochondrial Complex I.

In order to elucidate the electron transfer reaction of the enzyme complex, it is a prerequisite to identify the number and location of its redox components. It has been shown that the P. denitrificans NDH-1 contains one FMN (Yagi, 1986) and at least 5 EPR-visible iron-sulfur clusters analogous to those in bovine heart Complex I (Meinhardt et al., 1987). These iron-sulfur clusters are tentatively designated as N1a, N1b, N2, N3, and N4. The N1a and N1b are [2Fe-2S] clusters, while all others are [4Fe-4S] clusters. On the basis of sequence comparison of the deduced primary structures of the P. denitrificans NDH-1 subunits with their homologues regarding iron-sulfur cluster binding sites (Matsubara and Saeki, 1992), and of the resolution data of the bovine Complex I (Hatefi, 1985), NQO1, NQO2, NQO3, NQO9, and possibly NQO6 have been suggested to contain iron-sulfur clusters (Yagi, 1993; Yagi et al., 1993). Recently, Yano et al. (1994a) have succeeded in overexpressing the NQO2 (25 kDa) subunit in Escherichia coli. The expressed NQO2 subunit has been purified and characterized by UV-visible and EPR spectroscopy, which revealed that the NQO2 subunit ligates a single [2Fe-2S] cluster with rhombic EPR signal (g = 1.92, 1.95, and 2.00). Further spectroscopic studies including CD, MCD, and resonance Raman spectroscopy have confirmed that the [2Fe-2S] cluster in the NQO2 subunit is coordinated by cysteine residues (Crouse et al., 1994). More recently, site-directed mutagenesis studies have indicated that four conserved cysteine residues (Cys-96, -101, -137, and -141) coordinate the [2Fe-2S] cluster with a novel binding motif (Cx(4)CxCx(3)C) (Yano et al., 1994b).

The NQO3 (66 kDa) subunit, which is homologous to the 75-kDa subunit of the iron-sulfur (IP) subcomplex of Complex I, is also a putative iron-sulfur cluster ligating subunit. Therefore, we attempted to express the NQO3 subunit in E. coli in hopes that the NQO3 subunit might be natively expressed, as was the NQO2 subunit, and might better lend itself to a study of the properties of its iron-sulfur cluster(s). In the present paper we report the characterization of the P. denitrificans NQO3 subunit expressed in E. coli using pET11a expression vector. The expressed subunit was found predominantly in the cytoplasm and contains iron-sulfur clusters. The subunit was purified and then subjected to chemical analysis, UV-visible and EPR measurements to characterize the iron-sulfur clusters. We found that the NQO3 subunit contains at least one [2Fe-2S] and one [4Fe-4S] cluster. The assignment of these iron-sulfur clusters to a specific electron-transfer components of NDH-1 and possible functional role of the NQO3 (66 kDa) subunit are discussed.


EXPERIMENTAL PROCEDURES

Construction of Expression Vector

A full-length NQO3 gene was constructed from two plasmids, pXT-1 and pXT-2, as follows: a PstI/SphI fragment encoding the C-terminal region of NQO3 subunit was taken from pXT-2 and ligated into PstI/SphI cleaved pTZ18U. The plasmid thus obtained was designated pTZ18(NQO3c). In addition, a PstI fragment encoding the remaining N-terminal region of NQO3 subunit was cleaved from pXT-1 and ligated into PstI cleaved pTZ18(NQO3c). The proper construct was confirmed by cleavage with several restriction enzymes and finally by sequencing around the PstI ligation site (Sanger et al., 1977). The resulting construct was designated pTZ18U(NQO3). An oligonucleotide 5`-GATCTGCCATATGGTCTTCCCTT-3` (the italic and underlined bases were altered from the P. denitrificans DNA for the mutation) was designed in order to generate an NdeI site at the protein initiation codon. The mutagenesis was performed using the Bio-Rad in vitro mutagenesis kit based on the method of Kunkel et al.(1987). The mutation was confirmed by DNA sequence analysis of the region surrounding the initiation site. In order to avoid sequencing of the entire region, the BglII/BamHI fragment of the mutated plasmid was replaced with the BglII/BamHI fragment from pTZ18U(NQO3) plasmid and the resulting plasmid was designated pTZ18U(NQO3-NdeI). The NdeI/BamHI fragment from pTZ18U(NQO3-NdeI) was ligated in pET11a expression vector cleaved with NdeI/BamHI. The final construct was designated pET11a(NQO3) and was used for expression of the NQO3 subunit in E. coli.

Expression of NQO3 (66 kDa) Subunit

Expression of intact NQO3 subunit is highly dependent on the following conditions: bacterial strain, medium content, growth temperature, and induction time. Temperature is one of the key factors in obtaining the soluble intact products. In addition, the iron-sulfur cluster content of the expressed subunits is significantly affected by timing of induction with IPTG. The optimal expression procedure is as follows. Competent E. coli strain BL21(DE3)pLysS was transformed with pET11a(NQO3) and spread onto a 2xYT agar plate containing 50 µg/ml carbenicillin. A well isolated colony was picked and used to inoculate 10 ml of 2xYT medium containing 100 µg/ml ampicillin and cultivated at 37 °C to the stationary phase. The culture was used to inoculate 500 ml of TB medium containing 100 µg/ml ampicillin, 100 µg/ml ammonium Fe(II) citrate, and 100 µM sodium sulfide. Cells were grown at 25 °C until absorbance (at 600 nm) of the culture reached approximately 0.1. IPTG was then added to 0.4 mM final concentration and the cells were further grown for 18 h at 25 °C. The cells were harvested by centrifugation at 6,000 rpm for 10 min in a GSA rotor. The cell precipitates were suspended in 50 mM Tris-HCl buffer (pH 8.5) containing 1.0 mM EDTA, 0.1 mM PMSF, 1 mM DTT, and 150 mM NaCl to 20% (w/v). The cell suspensions were rapidly frozen in liquid nitrogen, and stored at -20 °C until use.

Purification of NQO3 (66 kDa) Subunit

The cell suspension was freeze-thawed twice using liquid nitrogen and a water bath at 30 °C and then sonicated for a few minutes on ice until its viscosity was reduced. After that, all procedures were performed at 4 °C. The suspension was treated twice with Parr cell disruption bomb at >1,000 kg/cm^2. The suspension was centrifuged at 6,000 rpm for 10 min in SS34 rotor in a Sorvall centrifuge to remove unbroken cells. The resulting supernatant was further centrifuged at 50,000 rpm for 30 min in a 60Ti rotor to separate soluble and membrane fractions. The supernatant was transferred into a beaker and the protein concentration was adjusted to approximately 5 mg/ml with buffer A (10 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.1 mM PMSF, and 1 mM DTT) plus 150 mM NaCl. The suspension was fractionated with (NH(4))(2)SO(4), and the fraction precipitating between 30 and 38% (NH(4))(2)SO(4) saturation was collected at 10,000 rpm for 15 min in SS34 rotor. The dark brown precipitate was suspended in 10 ml of buffer A and dialyzed against 1 liter of buffer A for 3 h. The dialyzed solution was applied on a DEAE-Toyopearl column (3.0 15 cm) equilibrated with buffer A. The column was washed with 10 column volumes of buffer A. The protein was eluted with a linear gradient of 0-0.5 M NaCl in buffer A. The fractions containing the NQO3 subunit were collected and dialyzed against 1 liter of buffer A for 3 h. The solution was applied on a QAE-Toyopearl column (3.0 10 cm) equilibrated with buffer A. After washing the column with 10 column volumes of buffer A followed by 10 column volumes of buffer A containing 0.05 M NaCl, the adsorbed proteins were eluted with a linear 400-ml gradient of 0.05-0.7 M NaCl in buffer A. The fractions containing the NQO3 subunit were combined and concentrated by Amicon Centriprep-30.

Gel Filtration Analysis

The purified NQO3 subunit (0.5 mg) was applied to a gel filtration column (Bio-Rad A-5m, 1.0 45 cm) equilibrated with 10 mM Tris-HCl buffer (pH 8.5), containing 1 mM DTT and 300 mM NaCl and eluted at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm with Pharmacia LKB Uvicord SII monitor. Molecular size standards used were beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (68 kDa), and ovalbumin (43 kDa).

Sequence Analysis

GCG software programs were used to analyze the amino acid sequence (Devereux et al., 1984). Sequence comparison of the polypeptides were conducted with BESTFIT and PILEUP programs. The FASTA and PROFILESEARCH programs were used to search for homology with the GenBank/EMBL sequence data bases. Homology search was also carried out by using the BLAST program running at the National Center for Biotechnology Information (Altschul et al., 1990).

EPR Spectroscopy

EPR measurements were conducted with a Bruker ESP300 E spectrometer operating at X-band (9.2 GHz). The sample temperature was controlled with an Oxford instrument ESR-9 helium flow cryostat. The magnetic field was calibrated using a strong or a weak pitch standard. Spin quantitations were conducted under non-power saturated conditions using 0.5 mM Cu-EDTA as a standard. The transition-probability corrections were made according to Aasa and Vnng(1975). EPR spectral simulations were performed as described by Blum and Ohnishi(1980) assuming Gaussian line shapes and no hyperfine interactions. Potentiometric redox titrations were done according to Dutton(1978).

Other Analytical Procedures

UV-visible absorption spectra were recorded on an SLM-Aminco DW-2000 spectrophotometer. Protein was estimated by the methods of Lowry et al.(1951) and Biuret in the presence of sodium deoxycholate (1 mg/ml) (Gornall et al., 1949). SDS-polyacrylamide gel electrophoresis was carried out by the modified method of Laemmli(1970). Immunoblotting was conducted as described (Han et al., 1988, 1989; Hekman and Hatefi, 1991; Hekman et al., 1991). Non-heme iron and acid labile sulfide were determined according to Doeg and Ziegler(1962) and Forgo and Popowski(1949), respectively. Amino acid composition (Yagi and Dinh, 1990), amino acid sequence (Matsudaira, 1987), and DNA sequences (Sanger et al., 1977) were determined according to the references cited. Any variations from the procedures and other details are described in the figure legends.

Materials

Acrylamide, N,N`-methylenebis(acrylamide), SDS, prestained low-range marker proteins, and Coomassie Brilliant Blue R-250 were from Bio-Rad; DNA sequencing kit was from U. S. Biochemical Corp; [alpha-S]thio-dATP was from Amersham; alkaline phosphatase-conjugating affinity purified antibodies to rabbit IgG were from Calbiochem; expression vectors and E. coli strain were from Novagen; carbenicillin was from ICN. The antiserum against the bovine 75-kDa subunit was a kind gift from Dr. Maureen G. Price (Rice University, Houston).


RESULTS

Expression of the NQO3 (66 kDa) Subunit in E. coli

We expressed the P. denitrificans NQO3 subunit as a non-fused protein in E. coli cells with pET11a vector (see ``Experimental Procedures''). The expression of intact NQO3 subunit is highly dependent on the growth conditions. When the expression is induced by the addition of IPTG at 37 °C, the NQO3 subunit is overexpressed as inclusion bodies. However, when the growth temperature was decreased to 25 °C, the subunit remained in the cytoplasmic phase. A similar effect was also observed previously in the case of the P. denitrificans NQO2 subunit (Yano et al., 1994a). These phenomena could be explained by the fact that bacterial cells grow slower at lower temperatures so that the expressed product may be given enough time for proper folding and cofactor incorporation. We have tried to express the NQO3 subunit in several different forms: fused-protein with His Tag sequence in the N terminus or C terminus, and in truncated forms with different polypeptide length (data not shown). However, in all of these attempts we failed to obtain comparable amounts of soluble products, and inclusion bodies were formed even at lower culture temperatures. Moreover, the iron-sulfur cluster content of the products in the soluble fraction was very low (data not shown). Probably, a full-length and non-fused form of the NQO3 subunit is essential for the proper polypeptide folding and cofactor incorporation. Fig. 1shows typical results of expression of NQO3 subunit in E. coli under optimal growth conditions. The overproduced NQO3 subunit constitutes up to 10% of the total soluble proteins in E. coli (Fig. 1A). The expressed product is readily recognized by anti-bovine 75-kDa antibody (Fig. 1B), which has been previously shown to cross-react with the NQO3 subunit of the P. denitrificans NDH-1 enzyme complex (Xu et al., 1992a).


Figure 1: Localization of the expressed NQO3 (66 kDa) subunit. The Coomassie Brilliant Blue-stained SDS-polyacrylamide gel (panel A) and immunoblotting (Panel B). The lanes contains cell lysate of E. coli pET(NQO3) (12 µg of protein for lane A1 and 1.2 µg for lane B1), soluble fraction (7.5 µg of protein for lane A2 and 0.75 µg for lane B2), and membrane fraction (2.1 µg of protein for lane A3 and 0.21 µg for lane B3). Immunoblotting was carried out using affinity-purified antibody against the bovine 75-kDa subunit and alkaline phosphate-conjugated anti-rabbit IgG antibody as described (Han et al., 1988, 1989; Hekman et al., 1991). The numbers at the left indicate the molecular mass in kilodalton and the arrows at the right indicate the expressed NQO3 subunit.



The expressed NQO3 subunit is found mainly in the soluble fraction, whereas the yield of the product in the membrane fraction is extremely low and could be detected only immunologically (Fig. 1). The NQO3 subunit was purified from the cytoplasmic fraction to nearly homogeneous form (see ``Experimental Procedures''), as revealed by SDS-polyacrylamide gel electrophoresis and by immunoblotting with antibody against bovine 75-kDa subunit (Fig. 2). Neither detergents nor chaotropic agents were required for the isolation. The yields of the purification steps are summarized in Table 1. Approximately 80 mg of purified protein was obtained from 100 g (wet weight) of E. coli cells. The N-terminal amino acid sequence of the purified expressed subunit is equivalent to that of the NQO3 subunit isolated from the P. denitrificans NDH-1 (A-D-L-R-K). The expressed subunit lacks the initial methionine which appears to be removed post-translationally in E. coli as well as in P. denitrificans cells (Xu et al., 1992a). The amino acid composition of the expressed NQO3 subunit (data not shown) is in reasonably good agreement with the composition deduced from the NQO3 gene sequence and also as determined by the amino acid analysis of the NQO3 subunit isolated from the P. denitrificans NDH-1 enzyme (Xu et al., 1992b). In addition, the identity of NQO3 gene product expressed in E. coli and the 66-kDa subunit isolated from P. denitrificans NDH-1 was confirmed by SDS-polyacrylamide gel electrophoresis.


Figure 2: SDS-PAGE and immunological analyses of samples from different stages of purification of the NQO3 (66 kDa) subunit. Panel A shows a Coomassie Brilliant Blue-stained SDS-polyacrylamide gel and panel B shows an immunoblotting using affinity-purified antibody against the bovine 75-kDa subunit. Lanes contain cell lysate of E. coli pET(NQO3) (10.6 µg of protein, lane A1; 1.1 µg of protein, lane B1), soluble fraction (8.2 µg of protein, lane A2; 0.8 µg of protein, lane B2), (NH(4))(2)SO(4) fraction (30-38% saturation) (3.4 µg of protein, lane A3), after DEAE-Toyopearl chromatography (1.4 µg of protein, lane A4), and after QAE-Toyopearl chromatography (1.0 µg of protein, lane A5; 0.1 µg of protein, lane B5). The numbers at the left indicate the molecular mass in kilodalton and the arrows at the right indicate the NQO3 subunit.





In order to examine whether the soluble NQO3 subunit exists as a monomer or an oligomer in solution without detergents, we estimated the apparent molecular size of the purified NQO3 subunit by gel filtration chromatography. The apparent molecular size of the NQO3 subunit was determined as approximately 140 ± 10 kDa, indicating that the soluble NQO3 subunits form dimers under the conditions used. No discernible amounts of more aggregated structures could be detected from the elution profile (data not shown).

The cytoplasmic fraction and (NH(4))(2)SO(4) precipitate prepared from E. coli cells with pET11a(NQO3) plasmid are dark-brown. This brown color becomes more distinct during the subsequent purification steps of DEAE- and QAE-Toyopearl column chromatography, suggesting that the expressed NQO3 subunit contains iron-sulfur clusters. We have determined the amount of non-heme iron and acid-labile sulfide in the preparations at different purification steps as shown in Table 1. After (NH(4))(2)SO(4) fractionation, the non-heme iron and acid-labile sulfide contents were almost equal. It should be noted that the supernatant prepared from E. coli cells harboring pET11a expression plasmid without the NQO3 gene contains much lower amounts of non-heme iron and acid-labile sulfide and shows no EPR signals characteristic of iron-sulfur clusters (data not shown). It seems likely that some part of iron-sulfur clusters in the expressed NQO3 subunit are lost during purification. As described below, we found that the [4Fe-4S] cluster in the expressed NQO3 subunit is not very stable and some of the clusters are converted to [3Fe-4S] clusters under aerobic condition.

Fig. 3shows the absorption spectra of the oxidized and reduced form of the expressed NQO3 subunit purified from the cytoplasmic fraction. The purified subunit shows broad absorption peaks at 320 and 417 nm in the oxidized form. In addition, it shows small shoulders around 460 and 550 nm. When the subunit is reduced by adding neutralized dithionite solution (5 mM final concentration), the color is bleached in the entire visible region. In the reduced form, a small broad peak is detected around at 550 nm. These results indicated that the expressed NQO3 subunit contains reducible iron-sulfur clusters in the molecule.


Figure 3: Optical absorption spectra of the purified NQO3 (66 kDa) subunit. The purified NQO3 subunit was diluted in 20 mM Tris-HCl (pH 8.5) containing 2.0 mM DTT and 1.0 mM EDTA and 0.1 mM PMSF to 1.25 mg/ml. The spectra were recorded at room temperature in the oxidized form (line A) and in the reduced form (line B). The sample was reduced by 5 mM dithionite (pH 7.5). The difference spectrum (oxidized form minus reduced form) is shown in the inset.



EPR Study of the Expressed NQO3 (66 kDa) Subunit

In order to determine the type and number of iron-sulfur clusters in the expressed NQO3 subunit, EPR measurements were carried out. Because of some loss of iron-sulfur clusters in the expressed NQO3 subunit during the last purification steps, we have used for EPR measurements the preparation after (NH(4))(2)SO(4) fractionation and DEAE-Toyopearl ion-exchange chromatography. In the reduced NQO3 subunit we have detected EPR signals from two distinct species of iron-sulfur clusters with different spin-relaxation behaviors. One species exhibits a spectrum of axial symmetry, and is detectable at sample temperatures as high as 77 K, indicating that it is a binuclear [2Fe-2S] cluster (Fig. 4A, solid line). The other shows a rhombic EPR spectrum arising from a rapidly relaxing species, most likely [4Fe-4S] cluster (Fig. 4B, solid line). Table 2summarizes the half-saturation parameters of EPR signals from these iron-sulfur clusters at various temperatures. The EPR spectra of the individual clusters can be simulated with reasonable fit using the following parameters: g = 1.934, 2.026, L = 1.8, 3.0 millitesla for the [2Fe-2S] cluster (Fig. 4A, dashed line); and g = 1.892, 1.928, and 2.063, L = 2.4, 1.55, and 1.75 millitesla for the [4Fe-4S] cluster (Fig. 4B, dashed lines).


Figure 4: EPR spectra of the iron-sulfur clusters of the expressed NQO3 (66 kDa) subunit of P. denitrificans NDH-1 (solid line) and their computer simulation (dashed line). The iron-sulfur clusters in the sample were potentiometrically poised to the E of -495 mV for [2Fe-2S] (A) and -464 mV for [4Fe-4S] clusters (B). EPR spectra were recorded at 35 K and microwave power at 10 mW for the [2Fe-2S] cluster (A); and 10K, 5 mW for the [4Fe-4S] cluster (B). Computer simulation of the clusters was conducted as described by Blum and Ohnishi(1980); parameters used were: g = 1.934, 1.934, and 2.026 and L = 17.9, 18.0, and 30.0 millitesla for the [2Fe-2S] cluster and g = 1.892, 1.928, and 2.063 and L = 24.0, 15.5, and 17.5 millitesla for the [4Fe-4S] cluster. Other EPR conditions: microwave frequency, 9.2506 GHz; modulation amplitude, 10 G; time constant, 64 ms.





To further characterize these two iron-sulfur clusters, a potentiometric redox titration was conducted. The Evalues of the two iron-sulfur clusters at pH 8.6 were determined to be -472 mV for the [2Fe-2S] cluster and -391 mV for the [4Fe-4S] cluster (Fig. 5). No additional EPR signals were detected down to E leq -600 mV. The relative stoichiometry of [2Fe-2S]:[4Fe-4S] clusters has been estimated as 1:0.84 on the basis of spin quantitation.


Figure 5: Potentiometric redox titration of the iron-sulfur clusters in the expressed NQO3 (66 kDa) subunit. The partially purified NQO3 subunit (after DEAE-Toyopearl chromatography) at 10 mg/ml in 50 mM Tris-HCl (pH 8.6), containing 1.0 mM DTT and 0.1 mM PMSF was titrated anaerobically at 15 °C in the presence of the following redox mediator dyes: 2-hydroxy-1,4-naphthoquinone, 1,2-naphthoquinone-4-sulfonate, 1,4-naphthoquinone-2-sulfonate, indigo disulfonate, indigo trisulfonate, indigo tetrasulfonate, safranin T, phenosafranin, neutral red, methyl viologen, and benzyl viologen (50 µM each). The reduction of the clusters was monitored by the signal amplitude at g = 1.93 at 35 K and 10-mW microwave power for the [2Fe-2S] cluster (▴) and at g = 1.89 signal at 10 K and 5-mW microwave power for the [4Fe-4S] cluster (▪). Other EPR conditions were as described in the legend to Fig. 4.



In the ferricyanide-oxidized NQO3 subunit, a characteristic EPR spectrum of an oxidized [3Fe-4S] iron-sulfur cluster was detected. Relative concentration of this cluster varied depending on preparation, ferricyanide concentration, and other experimental conditions. In order to clarify whether this cluster is an intrinsic component of the protein or it represents a product of oxidative deterioration of the tetranuclear cluster, we have studied the EPR spectra of the [3Fe-4S] and the [4Fe-4S] clusters after different incubation periods of the NQO3 subunit with 0.1 mM ferricyanide (Fig. 6, A and B). As shown in Fig. 6, the g = 2.01 signal of the [3Fe-4S] cluster increased with incubation time. Concomitantly, the content of the [4Fe-4S] cluster decreased, which was measured after reduction of the samples with dithionite (in the presence of redox mediators). These results strongly indicate that the [3Fe-4S] cluster observed in the preparation is not another component of the NQO3 subunit but rather originates from the oxidant-induced conversion of the native [4Fe-4S] cluster into a [3Fe-4S] cluster in a process similar to that described for a number of bacterial ferredoxins (Bell et al., 1982; Guigliarelli et al., 1985; Morgan et al., 1984; Thomson et al., 1981).


Figure 6: Ferricyanide-induced conversion of [4Fe-4S] to [3Fe-4S] cluster in the expressed NQO3 (66 kDa) subunit. EPR spectra of oxidized (A) and dithionite-reduced (B) purified NQO3 subunit before, 1 and 10 min after the addition of ferricyanide. C, changes in the concentration of [3Fe-4S] and [4Fe-4S] cluster during the time course of the incubation of NQO3 subunit with ferricyanide. Ferricyanide (0.5 mM) was added to the anaerobic solution of the purified NQO3 subunit (5 mg/ml) (50 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.1 mM PMSF, 40 µM methyl viologen, and 4 µM phenazine methosulfate), incubated in the EPR tube for the indicated time periods at +5 °C and frozen immediately before or after reduction with 10 mM dithionite. Spectra were recorded at 10 K. EPR conditions: microwave power, 5 mW; modulation amplitude, 8 G; microwave frequency, 9.46 GHz. The [3Fe-4S] and [4Fe-4S] clusters were monitored as g = 2.01 signal amplitude in the spectra of oxidized protein, and g = 1.89 signal amplitude of the dithionite-reduced samples, respectively. The small difference in the EPR line shape of [4Fe-4S] cluster is due to difference in the reduction level of the [2Fe-2S] cluster which is contributing to g(y) of the spectra. The relative large g = 2.00 free radical signal originates from the redox mediators.



Iron-sulfur cluster contents determined by EPR spin quantitation are approximately 0.8 mol of each cluster per subunit. Therefore, although the expressed NQO3 subunit is present as a homodimer, these results exclude the possibility that the iron-sulfur clusters are coordinated between two NQO3 subunit as exemplified by photosystem I (Golbeck, 1992) and nitrogenase iron protein (Georgiadis et al., 1992). Furthermore, these results also exclude the possibility that the binuclear and the tetranuclear clusters of the expressed NQO3 subunit share the ligand residues. Therefore, it is likely that the NQO3 (66 kDa) subunit houses at least one [2Fe-2S] and one [4Fe-4S] clusters in the molecule.


DISCUSSION

Based on the deduced primary structure of the P. denitrificans NQO3 subunit and its homologues, we anticipated that the NQO3 subunit carries plural iron-sulfur clusters (Xu et al., 1992a). Early EPR studies of the IP fragment as a whole, isolated from the bovine Complex I, containing the 75-kDa subunit (the homologue of the NQO3 subunit) together with six other water soluble polypeptides (Masui et al., 1991), showed two discernible axial-type EPR signals with largely different spin relaxation behavior, arising from at least one binuclear and one tetranuclear iron-sulfur clusters with g(z) values (2.02 and 2.06, respectively) fairly close to those described in the present paper. However, further chaotropic resolution of the IP fraction into smaller fractions resulted in considerable broadening of the spectra and to a loss of cluster-characteristic EPR properties (Ohnishi et al., 1985). In contrast, the present work revealed that the expressed single subunit of P. denitrificans NQO3 retained both iron-sulfur clusters. This supports the single subunit expression strategy for the assignment of the prosthetic groups to the NDH-1 subunits, as discussed in a series of previous papers (Yano et al., 1994a, 1994b; Crouse et al., 1994).

The NQO3 subunit contains 12 cysteine (Cys-26, -37, -48, -51, -66, -110, -113, -119, -158, -161, -164, and -208) and 2 histidine residues (His-39 and -106) (Xu et al., 1992a) that are conserved in complex I from various sources. Four cysteines (Cys-158, -161, -164, and -208) constitute a consensus sequence motif for the ligation of a [4Fe-4S] cluster (Cx(2)Cx(2)CxCP). Therefore, it seems likely that these cysteine residues coordinate the [4Fe-4S] cluster of the NQO3 subunit. The deduced primary sequence of the NQO3 subunit does not contain any cysteine sequence motifs for the [2Fe-2S] cluster ligation: the plant-type ferredoxins (Cx(4)Cx(2)CxC), hydroxylase-type ferredoxins (Cx(5)Cx(2)CxC), the NQO2 subunit (Cx(4)CxCx(3)C), or the Rieske iron-sulfur proteins (CxHxCx(2)H) (Matsubara and Saeki, 1992; Yano et al., 1994b; Gray and Daldal, 1995). To date, cysteine and histidine residues are the only residues which have been experimentally shown to coordinate [2Fe-2S] clusters in vivo. The ligand residues are most likely conserved in various organisms. It is thus conceivable that four out of the remaining 10 conserved residues (Cys-26, -37, -48, -51, -66, -110, -113, -119, His-39 and -106) are likely candidates for the [2Fe-2S] cluster ligation. Therefore, identification of the amino acid residues coordinating the [2Fe-2S] cluster of the NQO3 subunit would provide not only new information about the structure of the NQO3 subunit, but also a unique sequence motif for the [2Fe-2S] cluster ligation. This would provide additional support for the notion that NDH-1 and Complex I are the most elaborate of all known iron-sulfur proteins (Cammack, 1992; Johnson, 1994). In order to identify the ligand residues of the [2Fe-2S] and [4Fe-4S] clusters of the NQO3 subunit, site-directed mutagenesis experiments are in progress in our laboratories.

Generally speaking, 12 cysteines and 2 histidines in NQO3 provide enough room for the ligation of three iron-sulfur clusters. In addition, the amount of non-heme iron and acid-labile sulfide of the NQO3 subunit (8 mol of Fe/mol and 9 mol of S/mol) is approximately 1.8-fold higher than those based on the spin concentration by EPR measurements (4.8 mol of Fe/mol and 4.8 mol of S/mol). These facts suggest the possible existence of a third iron-sulfur cluster in the expressed NQO3 subunit. Bovine heart Complex I is also considered to contain EPR-silent iron-sulfur clusters (Hatefi, 1985). In order to verify this issue, further characterization of the NQO3 subunit by EPR, MCD, and resonance Raman spectroscopy is underway in collaboration with Dr. Michael K. Johnson's group.

Since the N3 cluster is most likely accommodated in the NQO1 (50 kDa) subunit (Ohnishi et al., 1981; Fecke et al., 1994; Sled' et al., 1994), the [4Fe-4S] cluster in the NQO3 subunit may be assigned to the N4 cluster. Two [2Fe-2S] clusters referred to as N1a and N1b are present in the P. denitrificans NDH-1 (Meinhardt et al., 1987). The N1a cluster exhibits an EPR spectrum with rhombic symmetry whereas the EPR spectrum of N1b cluster has an axial symmetry. Based on the EPR properties of the [2Fe-2S] clusters in the expressed NQO2 and NQO3 subunits, we could tentatively assign the N1a and N1b clusters to the NQO2 and NQO3 subunits, respectively. However, the midpoint redox potentials of the iron-sulfur clusters in the overexpressed subunits are considerably lowered in comparison with those in the complex. Since the subunits are solely expressed in E. coli in the absence of neighboring subunits, the environment of the iron-sulfur clusters might be different from that in the native enzyme complex. The midpoint redox potential of iron-sulfur clusters is especially sensitive to slight changes in the vicinity of the clusters (e.g. accessibility of external solvent). Substantial lowering of the midpoint redox potentials of iron-sulfur clusters has also been reported for the resolved FP subcomplex of bovine Complex I (Ohnishi et al., 1985).

As described above, all iron-sulfur clusters seem to be located in the N-terminal region (residues 1-265) of the NQO3 subunit. It has been reported that the N-terminal region of the NQO3 subunit is structurally similar to the -subunit of Alcaligenes eutrophus NAD-reducing hydrogenase which contains 11 of 12 cysteine residues conserved among the NQO3 subunit and its homologues (Xu et al., 1992a). The -subunit is thought to bear iron-sulfur cluster(s) and to participate in wiring electrons from the hydrogenase part of beta-heterodimer subunits to the NADH-binding site of alpha-subunit (Schneider et al., 1984; Tran-Betcke et al., 1990). Taken together, it is conceivable that the N-terminal region (iron-sulfur cluster domain) of the NQO3 subunit may participate in electron transfer reaction.

The iron-sulfur cluster domain of the NQO3 subunit seems to interact with FP, most likely with the NQO1 subunit. The evidence has been provided by cross-linking experiments of bovine heart Complex I (Patel et al., 1988a, 1988b; Yamaguchi and Hatefi, 1993), which indicates that the 75-kDa subunit is cross-linked with the 51-kDa subunit of FP (NQO1 subunit homologue). In addition, presuming the topological similarity between the P. denitrificans NDH-1 and the bovine heart Complex I, it can be speculated that the NQO3 subunit plays a structural role in connecting FP and HP with other IP subunits. Price and Gomer(1989) have purified the 75-kDa subunit of Complex I from bovine ventricular myocardium using a method for the isolation of the cytoskeletal component desmin. The isolated 75-kDa subunit was found to polymerize into the 10-nm filamentous structure in vitro. On the basis of these results, these authors proposed that the 75-kDa subunit functions as a membrane-associated filamentous skeleton. It is probable that this characteristic results from the relatively hydrophobic C-terminal region (residues 266-673) of the NQO3 subunit which may be involved in the interaction of its hydrophilic section of the Complex I or NDH-1 with its hydrophobic section (HP subunits). Taken together, it is suggested that the NQO3 subunit acts as the linking protein among FP, IP, and HP. This hypothesis should be experimentally examined in the future.


FOOTNOTES

*
This work was supported by United States Public Health Science Grants R01GM33712 (to T. Y.) and R01GM30376 (to T. O.). Facilities of computer were supported by United States Public Health Science Grant M01RR00833 for the General Clinical Research Center. Synthetic oligonucleotides were, in part, supported by the Sam & Rose Stein Endowment Fund. This is publication 9109-MEM from the Scripps Research Institute, La Jolla, CA and University of Pennsylvania, Philadelphia, PA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^1
The abbreviations used are: Q, quinone; NDH-1, bacterial proton-translocating NADH-quinone oxidoreductase; NDH-2 bacterial NADH-quinone oxidoreductase lacking an energy coupling site; Complex I, mitochondrial proton-translocating NADH-ubiquinone oxidoreductase; FP, IP, and HP, respectively, flavoprotein fraction, iron-sulfur protein fraction, and hydrophobic protein fraction of bovine Complex I; EPR, electron paramagnetic resonance; CD, circular dichroism; MCD, magnetic circular dichroism; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; DTT, dithiothreitol; IPTG, isopropyl-D-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride; mW, milliwatt(s).


ACKNOWLEDGEMENTS

We thank Catherine Guffey and Daniel G. Owen for their excellent technical assistance, Drs. Saeko Takano and Akemi Matsuno-Yagi for stimulating discussion, and Drs. Youssef Hatefi and Cecilia Hgerhll for critical reading of the manuscript.


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