Localization and function of the membrane-bound riboflavin in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae

Abstract

The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na(+) across the bacterial membrane. The Na(+)-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na(+)-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na(+)-NQR is discussed.

FIGURE 1.

Plasmids for expression of NQR subcomplexes. Only regions of plasmids downstream of the arabinose promoters are shown. Restriction sites on the parent plasmid pNQR1 that were used for cloning of truncated expression plasmids (black arrows) and introduced restriction sites (gray arrows) are indicated. DNA fragments that were deleted from the corresponding parent plasmid are shown as striped bars.

FIGURE 2.

Thin layer chromatography of flavins in Na⁺-NQR. Flavin standards and purified Na⁺-NQR were spotted on the TLC plate, developed with 2-butanol:glacial acetic acid:water (2:1:1) and analyzed by UV illumination. Approximately 150 pmol of standard or protein complex (Na⁺-NQR) were applied per spot.

FIGURE 3.

Detection of NqrA, NqrB, and NqrC in membranes containing NQR subcomplexes. Washed membranes from V. cholerae Δnqr transformants expressing different sets of Nqr subunits were solubilized with SDS and membrane proteins (30 μg) were separated by SDS-PAGE. FMN covalently bound to subunits NqrB and NqrC was detected by in gel fluorography (A and B). NqrA was detected by Western blot analysis against its N-terminal His₆ tag (C).

FIGURE 4.

Ag⁺-sensitive NADH oxidation by membranes containing subcomplexes of Na⁺-NQR. The specific NADH oxidation activity of washed membrane vesicles from V. cholerae Δnqr transformants expressing different sets of Nqr subunits were determined in the absence of Ag⁺ (black bars) and in the presence of 1 μm AgNO₃ (gray bars). Ubiquinone-1 was used as electron acceptor. Membranes from V. cholerae Δnqr and from transformants expressing NqrA served as controls. Mean values and standard deviations from at least two measurements are shown.

FIGURE 5.

Flavin analysis of membranes containing NQR subcomplexes. Washed membrane vesicles (∼1 mg of protein) from V. cholerae Δnqr transformants expressing different sets of Nqr subunits were analyzed for flavins by HPLC. Elution of flavins was monitored at 450 nm. Elution times of standards (FAD, 120 pmol; FMN, 37 pmol; riboflavin, 56 pmol) are indicated.

FIGURE 6.

DDM binding to Na⁺-NQR. A, separation of 1 mg Na⁺-NQR on a 1-ml HiTrap ANX-FF column (GE-Healthcare) monitored from the absorbance at 280 nm. B, concentrations of protein (gray bars) and protein-bound DDM (black bars). The concentration of protein-bound DDM was obtained by subtracting the DDM concentration of the buffer (0.639 ± 0.015 mg ml⁻¹) from the DDM concentration of the eluate.

FIGURE 7.

Determination of the mass of holo-Na⁺-NQR by static light scattering. The analysis was performed online with Na⁺-NQR eluting from a Superdex 200 column. The UV absorbance signal (thin solid line) and the differential refractive index signal (thin dashed line) are shown. Na⁺-NQR eluted at 21.94 min. The calculated molar masses of the detergent fraction (thick light gray line, bottom), the protein fraction (thick dark gray line, middle), and the protein-detergent conjugate (thick black line, top) are shown.

FIGURE 8.

Sedimentation velocity analysis of Na⁺-NQR. A, sedimentation distributions of Na⁺-NQR monitored by absorption at 280 nm at different time points are shown as colored dots. The fits of the curves are shown as black lines. B, residuals between experimental data and fits. C, protein molecular mass distribution obtained from the fits in A.

FIGURE 9.

Sequence alignment of NqrB. Sequences of NqrB from Chlamydia pneumoniae (Q9Z8B6), Protochlamydia amoebophila (Q6MEH4), Shewanella oneidensis (Q8EID9), Porphyromonas gingivalis (Q7MT18), Rhodopirellula baltica (Q7UWS4), and V. cholerae (A6XUU9) were compared. The alignment shown here is a section from a larger alignment of 69 non-redundant sequences of NqrB homologues, which was constructed using the ClustalW algorithm implemented in the BioEdit software (Version 7.0.9.0) with default settings (see “Experimental Procedures” and supplemental Fig. S1). Identical residues and similar residues are underlaid with black and gray bars, respectively, and were derived from the alignment in supplemental Fig. S1 using an identity and similarity threshold of 85%. Black bars indicate transmembrane helices, gray bars indicate hydrophobic stretches, as predicted from a consensus model based on 11 different topology prediction algorithms (see “Experimental Procedures” and supplemental Fig. S2). The FMN phosphorylthreonine (Thr-236) is highlighted by an asterisk, and Gly-141, which was proposed to interact with the inhibitor korormicin (11), is marked by a rhomboid.

FIGURE 10.

Partial sequence alignment of NqrB and flavodoxin. Amino acid sequences of flavodoxin from D. vulgaris (P00323) and Methanosarcina acetivorans (Q8TMF9) in the regions of the flavin-binding loops were aligned with the corresponding region of NqrB from C. pneumoniae (Q9Z8B6) and V. cholerae (A6XUU9). Residues of flavodoxin that mediate side-chain contact to riboflavin, including the two aromatic residues of flavodoxin, which stack to the isoalloxazine ring, are marked by arrows. Thr-236 of NqrB, which carries the covalently linked FMN, is indicated by an asterisk. Black bar, proposed transmembrane helix; gray bar, hydrophobic stretch.