NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit.

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit. This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V. cholerae or Escherichia coli as expression hosts. NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)). EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type. The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster. The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex. The observed electron transfer NADH --> FAD --> [2Fe-2S] in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.

The ability to diminish the intracellular Na ϩ concentration by specific transporters is common to many organisms. The uphill transport of Na ϩ against an electrochemical potential catalyzed by Na ϩ /H ϩ antiporters is driven by the proton motive force (1). In addition, some bacteria and archaea possess Na ϩ pumps that directly couple an exergonic reaction to the endergonic transport of Na ϩ across the membrane (2). For example, the oxidation of NADH with quinone catalyzed by membranebound NADH dehydrogenases generates an electrochemical Na ϩ gradient that can be used to drive the uptake of nutrients. Two distinct classes of bacterial Na ϩ -translocating NADH dehydrogenases are known. Enterobacteria like Escherichia coli possess a Na ϩ -dependent NADH dehydrogenase that is homol-ogous to complex I of the mitochondrial respiratory chain (3)(4)(5). Another type of NADH-driven redox pump (called Na ϩ -NQR) 1 is found in marine bacteria like Vibrio alginolyticus or the human pathogen V. cholerae (6 -11). The Na ϩ -NQR is encoded by the nqr operon that comprises the structural genes nqrA-nqrF (12,13) encoding for six different subunits present in the complex (9). NqrF is the only subunit that contains four closely spaced cysteine residues 70 Cys-Xaa 5 -Cys-Xaa 2 -Cys-Xaa 31 -Cys 111 (V. cholerae numbering) required for the ligation of an Fe-S cluster. In addition, motifs for the binding of flavin and NADH were identified on NqrF (14). Subunits NqrB and NqrC each contain one FMN that is covalently linked to a threonine residue (15)(16)(17). Recently, riboflavin was identified as a component of the Na ϩ -NQR complex from V. cholerae (18). In summary, the known prosthetic groups of the Na ϩ -NQR are one non-covalently bound FAD, two covalently bound FMNs (on NqrB and NqrC, respectively), one riboflavin, one Fe-S cluster (on NqrF), and ubiquinone-8 (6,19). The central question is how electron transfer from NADH to the substrate quinone proceeds and how this redox reaction is coupled to the translocation of Na ϩ by the Na ϩ -NQR. Here we demonstrate that the NqrF subunit catalyzes the initial electron transfer reactions NADH 3 FAD 3 [2Fe-2S] in the Na ϩ -NQR complex.

EXPERIMENTAL PROCEDURES
Construction of Expression Vectors-V. cholerae O395 N1 (20) served as a source of genomic DNA for PCR cloning of nqrF constructs. Cloning was carried out in E. coli DH5␣ using standard techniques (21). Gene sequences encoding the shortened NqrF subunit, its Fe-S domain, or flavin domain were amplified from chromosomal DNA by PCR. The forward primers for amplification of sequences encoding a truncated NqrF subunit or its Fe-S domain were designed to excise bp 7-75 of nqrF. Hereby, amino acids Thr 3 -Ala 25 including the predicted single N-terminal transmembrane helix (Val 8 -Ala 25 ) of the NqrF subunit were removed, and soluble variants of NqrF (NqrFЈ) and its Fe-S domain were produced (see Fig. 1). Primer sequences and restriction sites are given in Table I. The 3Ј-ends of the reverse primers for NqrFЈ and the flavin domain construct were homologous to sequences downstream of the stop codon of nqrF. PCR amplification by Pfu polymerase (Stratagene) was carried out as described by the supplier with an annealing temperature of 56°C and an amplification time of 2 min and 40 s. PCR products encoding for NqrFЈ or the flavin domain were digested with NdeI and EcoRI and ligated into the arabinose-inducible expression vector pEC422 (22), yielding pNF3 and pFNF8. The PCR fragment encoding the Fe-S domain was digested by NdeI and XhoI and inserted into pET-16b (Novagene) to give pFS224. The translation product of pFS224 has a C-terminal extension of 21 amino acids, and the stop codon is conferred by the vector. All three constructs add N-terminal His tags to the target proteins consisting of six histidine residues in the case of the NqrFЈ and its flavin domain and ten histidine residues in the case of the Fe-S domain. The expression vectors pNF3 or pFNF8 were transformed into V. cholerae as described in Ref. 23. E. coli BL21 (DE3) (Stratagene) was transformed with pFS224. The cloned gene fragments were sequenced, and in the case of the fragments encoding the NqrFЈ and the flavin domain the fragments were found to be identical to the corresponding genomic sequence of V. cholerae El Tor N16961 (Gen-Bank TM accession number AAF95434) (24). The cloned gene fragment encoding the Fe-S domain contained a silent G3 A mutation at bp 273 (nqrF numbering). The identity of the Fe-S and the flavin domains with the predicted polypeptides derived from nqrF from V. cholerae El Tor N16961 was further supported by matrix-assisted laser desorption ionization-mass spectrometry. The observed mass was 17895 Da (calculated, 17873 Da) for the Fe-S domain and 33983 Da for the flavin domain (calculated, 34047 Da). NqrFЈ, the FAD, and the Fe-S domain showed the expected N-terminal sequences.
Cultivation of Bacteria-All strains were cultivated aerobically in Luria-Bertani (LB) medium (21). V. cholerae O395 N1 was cultivated in LB medium supplemented with 10 mM glucose and 50 mM potassium phosphate buffer, pH 7.3, in the presence of 50 g ml Ϫ1 streptomycin and 200 g ml Ϫ1 ampicillin. For the production of NqrFЈ or the flavin domain, 10 liters of medium were inoculated with 250 ml of V. cholerae O395 N1 transformed with pNF3 or pFNF8. The cells were grown in a 12-liter fermenter (Bioengineering AG) at 37°C to an A 600 nm of 0.8 or 1.0, respectively. Expression was induced by adding 10 mM L-arabinose. Four hours after induction (A 600 nm of ϳ2) the bacteria were harvested by centrifugation. For the production of the Fe-S domain 150 liters of LB medium containing 100 g ml Ϫ1 ampicillin were inoculated with 4 liters of E. coli BL21 (DE3)-pFS224 in a bioreactor (Bioengineering AG). The cells were grown at 37°C to an A 600 nm of 1.1. After the addition of 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside, growth was continued at 30°C overnight. The cells were harvested by centrifugation (d3A 112M-4 centrifuge, Loher) and washed with 10 mM Tris/HCl, pH 7.4, 0.3 M NaCl. Concentrated cell suspensions were frozen in liquid nitrogen and stored at Ϫ80°C. In Vitro Reconstitution of the Fe-S Cluster-Insertion of the Fe-S cluster was performed by means of the cysteine desulfurase NifS from Azotobacter vinelandii (25) under exclusion of oxygen. NifS (50 g/ml) was added to the Fe-S domain (Ͻ0.5 mM in 50 mM Tris/HCl, pH 7.8, containing 10 mol of (NH 4 ) 2 Fe(SO 4 ) 2 /mol of Fe-S domain and 10 mM dithiothreitol), and the reaction was started by the addition of 2 mM cysteine, pH 7.5, acting as sulfide donor. After 45 min, the reconstituted Fe-S domain was purified from the reaction mixture by affinity chromatography on the Ni-NTA-agarose column.

Purification of Recombinant NqrFЈ and Its Flavin and Fe-S Do
Reduction and Determination of Midpoint Potential-The reduction of NqrFЈ, the FAD, or the Fe-S domain was followed spectrophotometrically under exclusion of oxygen in cuvettes sealed with a rubber stopper. NADH or sodium dithionite were added in the glove box or with gas-tight syringes. The standard reduction potential of the FAD of the flavin domain was determined by the xanthine/xanthine oxidase method (26) in 100 mM Tris/HCl, pH 7.5, using 10 -20 M phenosafranin (E m ϭ Ϫ266 mV at pH 7.5, n ϭ 2) as suitable redox indicator. Benzyl viologen (E m ϭ Ϫ359 mV, pH 7.5, n ϭ 2) and methyl viologen (E m ϭ Ϫ440 mV, pH 7.5, n ϭ 2) were used as redox mediators (27) at final concentrations of 1.8 M each. The redox state of FAD or phenosafranin was monitored at 404 or 518 nm, respectively. The Nernst coefficient was determined from the slope of the line ln(FAD ox /FAD red ) versus ln(PS ox /PS red ). The difference between the E m of FAD in the flavin domain and the E m of phenosafranin was calculated from the vertical intercept of the line.
Analytical Methods-Protein was determined by the bicinchoninic acid method using the reagent from Pierce (28) or by the microbiuret method (29) preceded by trichloroacetic acid precipitation. Bovine serum albumin served as the standard. The concentration of active NqrFЈ and the flavin domain was estimated from the content of FAD after extraction. The protein concentration of the flavin domain and the Fe-S domain was standardized by UV spectroscopy using the calculated extinction coefficients at 280 nm based on the content of aromatic amino acid residues of the apoproteins. The concentrations of NADH (⑀ 340 ϭ 6.22 mM Ϫ1 cm Ϫ1 ), ubiquinone-1 (⑀ 275 ϭ 13.7 mM Ϫ1 cm Ϫ1 in ethanol or ⑀ 275 ϭ 7.8 mM Ϫ1 cm Ϫ1 in H 2 O) (30) and FAD (⑀ 450 ϭ 11.3 mM Ϫ1 cm Ϫ1 ) (31) were calculated based on their absorption coefficients.
SDS-PAGE was performed with 12.5% polyacrylamide (32). The expression of the nqrF constructs was confirmed by Western blotting using an antibody directed against histidine tags (Tetra-His Antibody, Qiagen). Non-covalently bound flavins were extracted from protein and subjected to high pressure liquid chromatography analysis (33).
Iron was determined colorimetrically by the 3-(2-pyridyl)-5,6-bis(5- sulfo-2-furyl)-1,2,4-triazinedissodium salt trihydrate (ferene) complex (34). For the determination of acid-labile sulfur the methylene blue method (35) was applied. NADH dehydrogenase activity was assayed in 20 mM Tris/H 2 SO 4 , pH 7.5, containing 50 mM Na 2 SO 4 , 50 g/ml bovine serum albumin, and 0.1 ubiquinone-1 (Sigma). The reaction was started by the addition of NADH. The kinetics were followed at 25°C in the presence of oxygen using a dual wavelength/double beam recording spectrophotometer (UV-3000, Shimadzu) operating in the dual wavelength mode at 340 -370 nm (36). Prior to activity assays NqrFЈ and the flavin domain were diluted to an appropriate concentration in 20 mM Tris/H 2 SO 4 , pH 7.5, supplemented with 1 mg/ml bovine serum albumin. During the activity measurements, diluted NqrFЈ was kept on ice under the exclusion of oxygen.
Spectroscopy-UV-visible spectroscopy was performed with a Cary 50 spectrophotometer (Varian) or a UV-3000 dual wavelength/double beam recording spectrophotometer. X-band EPR spectra were recorded with an Elexsys E-500 spectrometer equipped with a helium flow cryostat (Oxford Instruments ESR 410), a NMR Gaussmeter, and a Hewlett Packard Frequency counter. Simulation of EPR spectra was carried out by means of the program EPR (37). Spin concentrations were determined by comparison of simulated spectra to a CuSO 4 standard (38). CD spectroscopy of the Fe-S domain was performed using a MOS-450 spectropolarimeter (Biologics) at a scan rate of 10 nm/s at 25°C. The cuvette was flushed with N 2 .
Model Building-The sequence of the NqrF subunit from V. cholerae was aligned with the sequences of the benzoate 1,2-dioxygenase reductase (BenC) (39) and the cytochrome b reductase fragment of nitrate reductase (40) using the program 3D-PSSM (41). These alignments were combined and verified using structural information of BenC (Protein Data Bank entry 1KRH), of cytochrome b reductase (1CNF), and of adrenodoxin (42) (1AYF). The resulting multiple sequence alignment was the basis for a three-dimensional model of NqrF generated by the program modeler (43).

Cofactor Content and Activity of NqrF-derived Polypeptides-
The N-terminal half of NqrF with its binding motif for an Fe-S cluster is related to ferredoxins, whereas the C-terminal half comprises motifs for the binding of flavin and NADH (Fig. 1). Three NqrF-derived polypeptides were produced, the NqrF subunit devoid of a putative N-terminal ␣-helix with a molecular mass of 44,596 Da (NqrFЈ) and its Fe-S and flavincarrying domains with molecular masses of 17,873 and 34 047 Da, respectively (Fig. 2). NqrFЈ contained 0.94 Ϯ 0.06 mol of FAD/mol (21.1 Ϯ 1.3 nmol of non-covalently bound FAD/mg). In addition, less than 3.2 nmol/mg FMN and 0.3 nmol of riboflavin/mg were present in NqrFЈ. The FAD domain contained 0.95 Ϯ 0.06 mol of non-covalently bound FAD/mol (29.6 Ϯ 1.9 nmol of FAD/mg). Again, small amounts of FMN (Ͻ2.8 nmol/ mg) and riboflavin (Ͻ0.2 nmol/mg) were also detected. These results demonstrate that the non-covalently bound FAD present in the Na ϩ ⅐NQR complex resides in the NqrF subunit. Both NqrFЈ and the flavin domain exhibited high NADH dehydrogenase activities with ubiquinone-1 as an artificial electron acceptor (up to 20,000 mol min Ϫ1 mg Ϫ1 ), depending on the FAD content of the enzyme specimens. The increase of enzymatic activity with increasing concentrations of NADH was very similar for NqrFЈ and the flavin domain, with half-maximal activities observed in the presence of 2-4 M NADH. A characteristic property of Na ϩ -NQR is its inhibition by silver ions (44) increase in absorption with typical maxima at 340, 410, 451, and 560 nm. The content of iron and acid-labile sulfide indicated that the reconstituted Fe-S domain harbors a 2Fe-2S cluster in accordance with its spectroscopic properties (see below). We observed a rapid degradation of the Fe-S cluster in NqrFЈ and the Fe-S domain during few minutes after exposure to air. The NqrFЈ domain lost its brown-yellow color typical for Fe-S-containing flavoproteins and turned yellow, whereas the Fe-S domain bleached completely. A loss of the Fe-S center during purification in the presence of oxygen was also observed with the Na ϩ -NQR complex from V. alginolyticus (10).

Midpoint Redox Potential of the FAD in the Flavin Domain-
The flavin domain exhibited an optical spectrum typical for oxidized flavoproteins with maxima at 396 and 454 nm and a shoulder at 480 nm (Fig. 3). Upon addition of substoichiometric amounts of NADH (10 M) to the flavin domain (14 M), the transient formation of a blue neutral flavosemiquinone with a characteristic absorbance in the range from 520 to 660 nm was observed (Fig. 3A). The flavosemiquinone was stable for 20 -30 min. From the difference in absorbance at 580 nm and the absorption coefficient of the neutral flavosemiquinone in flavodoxin (⑀ 580 ϭ 4.5 mM Ϫ1 cm Ϫ1 ) (45), a flavosemiquinone concentration of 4 M was calculated indicating that ϳ30% of the FAD cofactor in the flavin domain was in the one-electronreduced state under these conditions. The formation of the one-electron-reduced flavin from the obligate two-electron donor NADH is unexpected and might be the result of a comproportionation reaction between two FAD domains in the fully oxidized and the fully reduced state, respectively (46). Addition of excess NADH (73 M) to the FAD domain (30 M) resulted in the complete reduction of the FAD (Fig. 3B). The Na ϩ -NQR complex purified in the presence of oxygen stabilized a neutral flavosemiquinone in the as isolated state, whereas an anionic flavosemiquinone was observed in the Na ϩ -NQR after addition of excess dithionite (7,47). We did not detect radicals in the flavin domain as isolated or in the flavin domain treated with an excess of NADH by EPR spectroscopy. The midpoint redox potential of the FAD in the flavin domain was determined by  Spectroscopic Properties of the Fe-S Domain-The cluster binding motif Cys-Xaa 5 -Cys-Xaa 2 -Cys-Xaa n -Cys (with n ϭ 29 -37) found on NqrF from different organisms is characteristic of vertebrate (or hydroxylase)-type 2Fe ferredoxins (48). The spacer between the first and the second ligating cysteine comprises five amino acid residues in vertebrate-type ferredoxins but only four residues in plant-type ferredoxins. It seems likely that Cys 111 (V. cholerae numbering) acts as the fourth ligand to the Fe-S cluster in NqrF, because other cysteines that could act as ligands are not conserved in NqrFs from different organisms. The visible absorption and circular dichroism spectra of the Fe-S domain from the NqrF subunit resembled that of the 2Fe ferredoxins (49) (Fig. 4). EPR spectroscopy of the Fe-S domain treated with a 15-fold excess of dithionite revealed a nearly axial signal which was assigned to a mixed-valent (Fe(II)/Fe(III)) state of a 2Fe-2S cluster with characteristic g values of gЌ ϭ 1.938 and gʈ ϭ 2.020 obtained by a simulation of the spectrum (Fig. 5). Axial EPR signals are characteristic of vertebrate-type 2Fe ferredoxins (50), whereas the spectra of plant-type 2Fe ferredoxins reveal a rhombic distortion (51). The signal of the Fe-S domain was readily saturated and was optimally detected at 1-25 W (12 K). EPR spectroscopy is a tool to quantify paramagnetic species like Fe-S clusters that exhibit a spin 1/2 in the appropriate redox state. The amount of reduced Fe-S cluster in the dithionite-treated Fe-S domain was 38 M according to spin quantitation of the simulated spec-trum, or ϳ50% of the expected amount of 2Fe-2S cluster based on the content of Fe and acid-labile sulfide.
Redox Properties of NqrFЈ-The visible absorption spectrum of NqrFЈ as isolated showed characteristic absorbance maxima at 396 and 456 nm ascribed to FAD. Additional maxima at 340 nm and around 560 nm were assigned to the Fe-S cluster (Fig.  6). Upon addition of dithionite (15 M) to NqrFЈ (15 M), part of the FAD in NqrFЈ was converted to the one-electron-reduced state under the formation of a blue neutral flavosemiquinone with characteristic absorbances in the range from 520 to 660 nm. Further addition of dithionite (23 M final concentration) resulted in the complete reduction of the FAD in NqrFЈ (Fig.  6A). Formation of a blue neutral flavosemiquinone in NqrFЈ was also observed after the addition of substoichiometric amounts of NADH (Fig. 6B). Approximately 50% of the FAD cofactor in NqrFЈ was in the one-electron-reduced state under these conditions. In the presence of excess NADH, the flavosemiquinone was converted to FADH 2 , but reappeared after 5 min (data not shown). A redox titration of the FAD in NqrFЈ using phenosafranin as an indicator dye revealed a Nernst coefficient of 0.33. Apparently, the FAD in NqrFЈ did not undergo complete two-electron reduction under conditions where full reduction of the FAD in the flavin domain was observed. These results suggest that the FAD in NqrFЈ has a more negative overall midpoint potential than the FAD in the flavin domain. The EPR spectroscopic properties of the Fe-S cluster in NqrFЈ and its isolated Fe-S domain were compared. Upon addition of NADH or dithionite to NqrFЈ, a nearly axial signal appeared that was very similar to the signals attributed to the 2Fe-2S cluster in the Fe-S domain. In addition, a small radical signal with a characteristic g-value at 2.00 was detected (Fig. 7, top trace). This radical signal was assigned to the neutral flavosemiquinone also observed by VIS spectroscopy (Fig. 6). Reduction of NqrFЈ with excess dithionite resulted in the disappearance of the radical signal as monitored by EPR (Fig. 7, bottom trace). As observed with the Fe-S domain, the Fe-S cluster in NqrFЈ was optimally detected at 10 (12 K) or 2 W (50 K) but was readily saturated at increasing microwave power. The g values of the Fe-S cluster were obtained from a simulation of the EPR spectrum of the dithionite-reduced NqrFЈ with gЌ ϭ 1.935 and gʈ ϭ 2.018. The amount of reduced Fe-S cluster in the NADH-treated NqrFЈ was 1 M according to spin quantitation of the simulated spectrum or ϳ1% of the amount of NqrFЈ based on FAD content. This is considerably lower than the amount of Fe-S cluster detected in the dithionite-reduced NqrFЈ (9.5 M, corresponding to ϳ10% of the amount of NqrFЈ based on FAD content). These results indicate that the 2Fe-2S cluster in NqrFЈ was only partially reduced using NADH as the electron donor, whereas complete reduction was achieved by the addition of dithionite, which has a more negative midpoint potential than NADH. Obviously, the midpoint potential of the 2Fe-2S cluster in NqrFЈ is significantly lower than the E m ϭ Ϫ320 mV of the NADH/NAD ϩ redox couple.

DISCUSSION
The Na ϩ -NQR contains four flavins, one Fe-S center, and ubiquinone-8 as cofactors. These redox centers are likely to participate in the electron transfer from NADH to membranebound quinones coupled to the transport of Na ϩ against an electrochemical potential. The description of the individual redox centers is a prerequisite to elucidate the transport mechanism of Na ϩ -NQR. A role for flavosemiquinones during redoxdriven Na ϩ transport has been proposed for the Na ϩ -NQR complexes from V. cholerae (47) and V. harveyi (7). In both studies, a neutral flavosemiquinone was observed in the Na ϩ -NQR purified in the presence of oxygen. Upon the addition of a reductant, this neutral flavosemiquinone disappeared, and an anionic flavosemiquinone was detected. Here we demonstrate that the NqrF subunit represents the site of electron entry into the Na ϩ -NQR complex. The FAD in NqrF acts as a converter between the hydride donor, NADH, and the one-electron acceptor, the 2Fe-2S cluster, under transient formation of a neutral flavosemiquinone. The anionic flavosemiquinone identified in the Na ϩ -NQR (7, 47) must therefore arise from one of the covalently bound FMNs on NqrB and NqrC, or from the riboflavin that is also present in the complex (18).
Productive electron transfer from the FAD to the 2Fe-2S cluster in NqrF requires a spacing of 14 Å or less between these redox centers (52). The structural arrangement of the FAD and the 2Fe-2S cluster can be predicted from a model of NqrF (Fig.  8) derived from high-resolution structures of related enzymes. Sequence comparisons indicate that the NqrF subunit comprises two functional units. The N-terminal part contains the cysteine motif required for the binding of the Fe-S cluster, whereas the C-terminal part is related to NADP ϩ -ferredoxin reductases (FNRs) (53) and contains the NADH-and FADbinding domains. NADP ϩ -ferredoxin reductases accept electrons from ferredoxin and reduce NADP ϩ to NADPH. The NqrF subunit can be viewed as a fusion of a 2Fe ferredoxin with its redox partner, FNR. The structural prototype of a 2Fe ferredoxin and a NADH-oxidizing FAD domain arranged in a single enzyme is represented by BenC. Like NqrF, BenC contains a 2Fe-2S cluster and FAD as redox cofactors and stabilizes a neutral flavosemiquinone upon reduction by NADH (54). A comparison of BenC with the related enzymes phthalate dioxygenase reductase and FNR in complex with ferredoxin revealed that the flavin and 2Fe-2S cluster can be superimposed in all three structures (39) indicating that the arrangement of the redox cofactors is highly conserved in this family of NAD(P)H dehydrogenases. We used the high-resolution structures of BenC (39) and related proteins (40,42) to model the structure of the NqrF subunit of the Na ϩ -NQR from V. cholerae. Important amino acid residues that act as ligands or form hydrogen bonds to the cofactors in BenC are also present in NqrF. For example, residues Arg 156 , Tyr 158 , and Ser 159 , which form hydrogen bonds to the phosphate, the ribityl and the isoalloxazine moieties of the FAD in BenC are fully conserved in NqrF (Arg 210 , Tyr 212 , and Ser 213 ). A stacking interaction of the isoalloxazine ring with Phe 335 is observed in BenC suggesting a similar arrangement of Phe 406 in NqrF. The ferredoxin domain of NqrF contains the four conserved cysteines Cys 70 , Cys 76 , Cys 79 , and Cys 111 , which act as ligands to the 2Fe-2S cluster. The Fe-S cluster is probably held in position by Gly 77 and Gln 112 (NqrF numbering), which in BenC are hydrogen bonded to the acid-labile sulfide S-2 of the 2Fe-2S cluster and to FIG. 7. EPR spectra of NqrF. NqrFЈ was mixed with NADH or sodium dithionite under exclusion of oxygen at room temperature and frozen in liquid nitrogen after 30 min. Top trace, 115 M NqrFЈ reduced with 580 M NADH in 50 mM Tris/HCl, pH 7.5. EPR conditions: microwave frequency, 9.6304 GHz; microwave power, 0.002 mW; modulation amplitude, 0.5 millitesla; temperature, 50 K. Bottom trace, 50 M NqrFЈ reduced with an excess of sodium dithionite in 20 mM glycine, 40 mM Tris/HCl, pH 8.3. EPR conditions: microwave frequency, 9.6342 GHz; microwave power, 0.01 mW; modulation amplitude, 1.0 millitesla; temperature, 12 K. Note that the EPR signal of the NADH-reduced NqrFЈ (top trace) exhibits much lower intensities than the signal of the dithionite-reduced NqrFЈ (bottom trace), which is shown at a 10-fold-reduced scale.
FIG. 8. Structural prediction of the NqrF subunit of the Na ؉ -NQR from V. cholerae. The model is based on the high-resolution structure of the benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1 (39), which exhibits 23% overall sequence identity to NqrF. A, proposed architecture of NqrF. The polypeptides produced are the ferredoxin domain (magenta), the flavin domain (blue), and a soluble variant of the NqrF subunit comprising the ferredoxin and the flavin domain (NqrFЈ), which is devoid of the N-terminal helix (green). This hydrophobic helix is assumed to anchor the NqrF subunit to the membrane. B, model of the FAD and the 2Fe-2S cluster in NqrF. The distance between the C(8) methyl group on the isoalloxazine ring of the flavin and the closest iron atom is sufficiently short for electron transfer. The adenosine moiety of the FAD was omitted for clarity. The figure was prepared with the program DS ViewerPro 5.0 (accelrys.com). the sulfur atom of the fourth cysteine, respectively. In summary, the FAD and the 2Fe-2S cluster are likely to adopt very similar positions in NqrF and BenC. The model of NqrF suggests a distance from the C(8) methyl group of the isoalloxazine ring of FAD to the closest iron atom of the 2Fe-2S cluster of ϳ9 Å (Fig. 8). This spacing would allow efficient electron transfer between the two redox cofactors, which is in agreement with the experimental results. The redox chain NADH 3 FAD 3 2Fe-2S on the NqrF subunit represents the initial electron transfer pathway in the Na ϩ -NQR complex. From the Fe-S center, electron transfer could proceed to one of the flavins or to ubiquinone-8 present in the complex. Subunits NqrC and NqrF were copurified as a fragment of the Na ϩ -NQR from V. alginolyticus (9). It therefore seems likely that the covalently attached FMN on NqrC is located in close proximity to NqrF and acts as an electron acceptor for the Fe-S center. It will now be important to investigate whether the oxidation of the Fe-S center precedes the formation of the anionic flavosemiquinone in the Na ϩ -NQR complex and whether this anionic flavosemiquinone is located on the NqrC subunit.