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

PLoS One. 2015 Mar 3;10(3):e0118548. doi: 10.1371/journal.pone.0118548. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Calorimetry
  • Crystallography, X-Ray
  • Flavin Mononucleotide / chemistry
  • Flavin Mononucleotide / metabolism*
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Quinone Reductases / chemistry
  • Quinone Reductases / metabolism*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Sequence Alignment
  • Vibrio / enzymology*

Substances

  • Bacterial Proteins
  • Protein Subunits
  • Recombinant Fusion Proteins
  • Flavin Mononucleotide
  • sodium-translocating NADH-quinone reductase
  • Quinone Reductases

Grant support

The work was supported by Russian foundation for basic research (research projects 13-04-91320) and Helmholtz Association of German Research Centres (project ID: HRJRG-401) as well as the Russian state Program 5top100-programme for enhancing the competitiveness of MIPT among the world’s leading research and education centers of the Ministry of education and science. NqrC purification and flavinylation as well as studying of FMN binding properties of apoNqrC′ were supported by RSCF research project 14-14-00128. Crystallization, X-ray data collection and treatment were supported by RSCF research project 14-14-00995. The work was supported by the program “Chaires d’excellence” edition 2008 of ANR France and CEA(IBS) - HGF(FZJ) STC 5.1 specific agreement. Part of this work was supported by BMBF (PhoNa - Photonic Nanomaterials) and Russian Federal Target Program “Research and Development” (№ 14.587.21.0004, unique identifier RFMEFI58714X0004). The work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.