A Vertebrate-type Ferredoxin Domain in the Na+-translocating NADH Dehydrogenase from Vibrio cholerae*

The Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mössbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys70, Cys76, Cys79, and Cys111. Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.

Iron-sulfur proteins are present in all domains of living organisms where they exhibit diverse functions like electron transport, catalysis, and sensing in regulatory processes (1). In some NADH-oxidizing, respiratory complexes, Fe-S centers accept electrons from flavin cofactors in an overall exergonic reaction that results in the reduction of quinone. This electron transfer reaction drives the uphill transport of protons or Na ϩ across the inner membrane of mitochondria or bacteria. The NADH:quinone oxidoreductase (Na ϩ -NQR) 1 from the human pathogen Vibrio cholerae maintains an electrochemical Na ϩ gradient across the inner bacterial membrane, which strongly influences the production of virulence factors (2). The Na ϩ -NQR consists of six subunits, NqrA-F, and contains one Fe-S center, two covalently bound FMNs, one non-covalently bound FAD, one riboflavin, and ubiquinone-8 as prosthetic groups (3)(4)(5)(6)(7). The NqrF subunit of the Na ϩ -NQR complex is anchored to the inner membrane and displays a clearly defined domain structure. The N-terminal Fe-S domain harbors the [2Fe-2S] cluster, while the binding sites for the non-covalently bound FAD and NADH are located in the C-terminal domain of NqrF. The initial oxidation of NADH by the NqrF subunit results in the two-electron reduction of the FAD followed by one-electron transfer steps to the [2Fe-2S] cluster in the Fe-S domain (7). Here we study the electronic properties of the [2Fe-2S] cluster in the isolated Fe-S domain of NqrF. A comparison of its amino acid sequence with sequences of [2Fe-2S] ferredoxins from vertebrates and plants reveals that the Fe-S domain is related to ferredoxins of the vertebrate-type family. Vertebrate-type ferredoxins are soluble redox carriers that accept electrons from specific NADH:ferredoxin reductases and deliver them to enzymatic systems catalyzing the hydroxylation of various compounds like steroids or camphor (8). The Fe-S domain exhibits highest sequence similarity to vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters (ISC-type ferredoxins) (9). This is further supported by Mössbauer and magnetic circular dichroism (MCD) spectra of the Fe-S domain, which are reminiscent to ISC-type ferredoxins. Our finding that a vertebrate-type [2Fe-2S] cluster is an intrinsic redox cofactor of the Na ϩ -translocating NADH dehydrogenase adds a novel function in respiration to this class of Fe-S centers.

Preparation of the Fe-S Domain-The
Fe-S domain of the Na ϩ -NQR comprises the amino acids Met 1 -Phe 146 of subunit NqrF devoid of the hydrophobic residues Val 8 -Ala 25 , which are likely to anchor the NqrF subunit to the membrane. The molecular mass of the Fe-S domain including its N-terminal polyhistidine tag is 17,873 Da. Following NifSmediated in vitro reconstitution of the Fe-S cluster (10), the Fe-S domain was purified by nickel-nitrilotriacetic acid affinity chromatography to remove precipitated iron sulfides and the cysteine desulfurase, NifS (7).
Spectroscopy-For Mössbauer spectroscopy, the NifS-mediated reconstitution of the Fe-S cluster of the Fe-S domain (7) was carried out with the 57 Fe-enriched Mohr's salt, (NH 4 ) 2 57 Fe(SO 4 ) 2 ⅐6H 2 O, as source of redox-stable ferrous ions. The salt was obtained from metallic iron foil (95% 57 Fe) by adding a stoichiometric amount of 1 M H 2 SO 4 to 1.88 ϫ 10 Ϫ4 mol of metallic 57 Fe. The solution was heated until the iron foil was completely dissolved. The volume was kept constant by adding distilled water to compensate for evaporation. Subsequently, the solution was concentrated at 80°C until a thin crystal film appeared on its surface. Likewise, 1.88 ϫ 10 Ϫ4 mol of (NH 4 ) 2 SO 4 was dissolved in a few drops of distilled water and concentrated at 80°C until crystallization commenced. The two hot solutions were mixed and crystals of (NH 4 ) 2 57 Fe(SO 4 ) 2 ⅐6H 2 O formed overnight at room temperature. The mother liquor was removed and the dried crystals were stored in the anaerobic chamber until use. Mössbauer data were recorded with a spectrometer of the alternating constant-acceleration type equipped with a Variox Cryostat (Oxford Instruments). The minimum experi-mental line width was 0.24 mm s Ϫ1 (full width at half-height). The 57 Co/Rh source (1.8 GBq) was positioned at room temperature inside the gap of the magnet system at a zero-field position. Isomer shifts are quoted relative to iron metal at 300 K. For MCD spectroscopy, the Fe-S domain in 50 mM Tris/HCl, pH 7.8, 40 mM oxalate, 4 M 5-deazaflavin, and 50% (v/v) glycerol was irradiated with white light for 10 min in the MCD cell immediately prior to freezing in liquid nitrogen. The photochemical 5-deazaflavin/oxalate system generates electrons at very low potentials of Ϫ650 mV (11) required for the reduction of the Fe-S domain. MCD spectra were obtained at liquid He temperatures (1.8 -50.4 K) on a Jasco J-715 (200 -1060 nm) with an extended S-20 and S-1 photomultiplier tube (Hammamatsu). The J-500C spectrometer was equipped with an Oxford Instruments SM4-11 T superconducting magnet/cryostat capable of fields up to 11 T and temperatures down to 1.5 K.
Analytical Methods-Protein was determined by the microbiuret method (12) preceded by trichloroacetic acid precipitation. Bovine serum albumin served as standard. The concentration of the Fe-S domain was determined by the microbiuret method standardized by UV spectroscopy using the theoretical extinction coefficient at 280 nm, 6290 M Ϫ1 cm Ϫ1 , of the colorless Fe-S domain devoid of its Fe-S cluster. Iron was determined colorimetrically by the 3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazinedisodium salt trihydrate (ferene) complex (13). For the determination of acid-labile sulfur the methylene blue method (14) was applied.

RESULTS AND DISCUSSION
Visible absorption and EPR spectra of the Fe-S domain of the NqrF subunit are reminiscent of [2Fe-2S] ferredoxins of the vertebrate type, with a maximum around 540 nm in the visible spectrum and a nearly axial EPR signal with g ʈ Ϸ 2.020 and g ʈ Ϸ 1.938 in the reduced state. The EPR spectrum of the Fe-S domain was indistinguishable from the spectrum of the NqrF subunit, indicating a very similar structural environment of the cluster in the two proteins (7). Axial S ϭ 1/2 EPR signals are typical of [2Fe-2S] ϩ clusters in vertebrate-type ferredoxins (15), while [2Fe-2S] ϩ centers of plant-type ferredoxins exhibit rhombic S ϭ 1/2 resonances. Sequence comparisons were performed with the aim to assign the Fe-S domain to known classes of ferredoxins. The NqrF subunit is highly conserved among Na ϩ -NQRs of different organisms, especially among Vibrio sp. (identity Ͼ90%). Including NqrF of Chlamydia sp., which form a group apart from other identified NqrF sequences, reduces the consensus to 36% identity (Fig. 1). The [2Fe-2S] cluster in NqrF is coordinated by the conserved residues Cys 70 , Cys 76 , Cys 79 , and Cys 111 (16). The arrangement of these cysteine residues matches the C-X(5)-C-X(2)-C-ϫ(30 Ͻ n Ͻ 39)-C cluster binding motif characteristic of vertebratetype [2Fe-2S] ferredoxins with the conserved residues RLXCQ surrounding the fourth cysteine ligand (C111 in V. cholerae NqrF). In addition to the cluster binding motif, an important region conserved between NqrF and vertebrate-type ferredoxins is the ␤ strand from M119 to L123, corresponding to Met 103 -Val 107 in adrenodoxin, followed by a conserved proline (P124 in NqrF) (8). A BLAST search (17) was performed using only the Fe-S domain of NqrF from V. cholerae as the input sequence. The highest similarity of the Fe-S domain to a biochemically characterized 2Fe ferredoxin was found for the ISCrelated ferredoxin 5 from Aquifex aeolicus (21% identity and 34% similarity) (18), followed by the ISC-type ferredoxin Yah1p from Saccharomyces cerevisiae (19,20) and ferredoxin IV from Azotobacter vinelandii (21,22) (Fig. 1). Yah1p and FdIV are essential components of the ISC assembly machinery, a biochemical pathway for the formation of iron-sulfur proteins in eukaryotes and bacteria (9,23).
Mössbauer spectroscopy is a powerful tool to determine the number of irons per cluster, its oxidation state, and the type of ligands to the Fe atoms (24). The zero-field Mössbauer spectrum of the oxidized Fe-S domain of Na ϩ -NQR recorded at 80 K shows an asymmetric doublet as depicted in Fig. 2. The spectrum could be deconvoluted into three symmetric quadrupole doublets with Lorentzian line shape. The major component, with 80% relative intensity, has small quadrupole splitting ⌬E Q ϭ 0.61 mm/s and characteristic low isomer shift ␦ ϭ 0.283 mm/s, which is typical of ferric iron with tetrahedral sulfur coordination (18,25,26). Since the subspectrum does not show any indications of paramagnetic broadening, and moreover, the Fe-S domain in the as isolated state is EPR-silent, the major contribution of the Mössbauer subspectrum can be assigned to a diamagnetic Fe(III)-Fe(III) pair due to the presence of binuclear [2Fe-2S] 2ϩ clusters. This is further supported by the content of Fe and S 2Ϫ (1.61 Ϯ 0.28 and 1.31 Ϯ 0.11 mol/mol of Fe-S domain, respectively). Two further subcomponents are found in the Mössbauer spectrum with the following simulation parameters: ⌬E Q ϭ 1.142 mm/s and ␦ ϭ 0.436 mm/s, weight 8% (component 2) and ⌬E Q ϭ 2.469 mm/s and ␦ ϭ 1.342 mm/s, weight 12% (component 3). The distinct high isomer shift of component 3 is typical of hexa-coordinated Fe(II) with "hard" donor ligands. We assign it to remaining Fe(II) ions in the solution (hex-aquo complex or non-specifically bound iron), originating from (NH 4 ) 2 57 Fe(SO 4 ) 2 ⅐6 H 2 O, the iron source in the reconstitution assay. The Mössbauer parameters of the minor component 2 resemble those of the delocalized mixed valence Fe(2.5)/Fe(2.5) pair of a cubane 4Fe-4S cluster (1). However, the component cannot be clearly discriminated from high spin Fe(III) precipitates due to the presence of some nonspecifically bound iron oxide or hydroxide.
To further compare the [2Fe-2S] cluster in the Fe-S domain with other ferredoxins, we applied variable temperature magnetic circular dichroism (VTMCD) spectroscopy, a complementary approach to EPR for investigating the electronic properties of paramagnetic iron-sulfur clusters. In particular, VTMCD spectroscopy allows to distinguish between vertebrate-and plant-type [2Fe-2S] ϩ centers and provides a sensitive probe of the electronic structure of clusters with paramagnetic ground states (27). Each electronic transition from a spin degenerate ground state gives rise to positive or negative ab-sorption-shaped MCD bands that increase in intensity with decreasing temperature. These C-term features commonly dominate the MCD spectra of paramagnetic species. The VT-MCD spectrum of the photochemically reduced Fe-S domain (Fig. 3) reveals a negative band at 315 nm assigned to Fe(II)-S charge transitions and positive bands at 358, 409, 461, 554, 629, and 694 nm assigned to Fe(III)-S charge transitions. This pattern of negative and positive bands is strikingly similar to the MCD spectrum of the [2Fe-2S] cluster found in the ISCtype Fd IV (18) or in the central domain of the ISC scaffold protein NifU (28). The spectrum of the Fe-S domain is clearly distinct from the MCD spectra of plant-type ferredoxins, which exhibit a prominent positive band at 515 nm (29). A comparison of the MCD spectrum of the Fe-S domain with the spectrum of other vertebrate-type Fds like putidaredoxin (29) shows that the latter displays an additional band in the range between 409 and 461 nm, which is not present in the Fe-S domain. We conclude that the [2Fe-2S] cluster in the isolated Fe-S domain of the Na ϩ redox pump is very similar to the 2Fe cluster from vertebrate-type ferredoxins and among these is mostly related to the [2Fe-2S] cluster present in ISC-type ferredoxins. ISCtype Fds catalyze an essential step during the biogenesis of iron-sulfur clusters (9). The in vitro formation of Fe-S clusters with nuclearity Ͼ2 requires a reductive step (1). In vivo, this redox reaction is likely to be catalyzed by ISC-type ferredoxins and specific dehydrogenases, which act as electron carriers during ISC assembly (9,20,23,30). The NqrF subunit of the Na ϩ -translocating NADH:quinone oxidoreductase represents a model for studying electron transfer from NADH to an ironsulfur cluster that is very similar to the [2Fe-2S] center found in ISC-type ferredoxins.