Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase from Vibrio harveyi

Abstract

Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium.

Fig 1. Noncovalent binding of free FMN to apoNqrC'

studied by the flavin and the protein fluorescence shown on the left and right panels, respectively. (a) Fluorescence of 0.35 μM free FMN (thick blue line) and 0.35 μM holoNqrC' (thick black line). The thin lines represent titration of fluorescence of free FMN (0.35 μM) by increasing amounts of apoNqrC' (apoNqrC' concentrations varied from 0.08 to 45 μM). (b) Tryptophan fluorescence of 0.32 μM apoNqrC' (thick blue line) and 0.32 μM holoNqrC' (thick black line). The red and green thin lines represent fluorescence of 0.32 μM apoNqrC' in the presence of 0.32 and 0.64 μM of free FMN, respectively.

Fig 2. Crystal packing of the holoNqrC' protein.

FMN residue is shown in orange. (a) and (b)—along a and c axis, respectively.

Fig 3. HoloNqrC' structure.

(a) and (b)—Overall view with 90°-rotation. Different secondary structure elements are shown in colors: β-sheets in yellow, α-helices in cyan, 3₁₀-helix in green. β-strands and helices are designated with numbers and Latin latters, respectively. Secondary structure was assigned with DSSP [29].

Fig 4. FMN binding site of holoNqrC'.

The protein is shown as a space-filling model at (a). H-bonds stabilizing the conformation of FMN residue are shown at (b). The intensity of orange color represents the 4 levels of amino acid conservation in agreement with Fig. 5. Green color represents nonconservative amino acids.

Fig 5. Sequence alignment of the NqrC subunits of NQR from different bacteria (V. harveyi (Vh_NqrC), Yersinia pestis (Yp_NqrC), Haemophilus influenza (Hi_NqrC), Pasteurella multocida (Pm_NqrC), Neisseria meningitidis (Nm_NqrC) and Pseudomonas aeroginosa (Pa_NqrC)) as well as paralogous RnfG subunits of the RNF complex from E. coli (Ec_RnfG) and V. cholerae (Vc_RnfG).

The intensity of orange color represents the 4 levels of amino acid conservation (calculated in [30]).

Fig 6. Ellipsoids of atomic-displacement parameters of the FMN residue in NqrC' drawn at 50% probability.

Numeration of the isoalloxazine atoms is shown according to the IUPAC nomenclature.