Recombinant Vibrio harveyi Na (+)-translocating NADH-quinone reductase subunit C (nqrC)

What is the structural organization of the NqrC subunit within the Na⁺-NQR complex?

NqrC is one of six subunits (NqrA-F) that compose the Na⁺-translocating NADH:quinone oxidoreductase complex. Based on cryo-EM studies, the NqrC subunit is positioned within the complex in a way that allows it to participate in electron transfer. The subunit contains a covalently attached FMN cofactor that serves as a key component in the electron transport chain of the complex .

Recent structural investigations have revealed that the distances between several pairs of redox cofactors, including those in NqrC, are shorter than previously thought based on crystallographic studies, which is more consistent with physiologically relevant electron transfer rates . The arrangement of NqrC relative to other subunits facilitates electron movement through the complex.

What cofactors are associated with the NqrC subunit and how are they identified?

The NqrC subunit has a covalently attached FMN (flavin mononucleotide) cofactor. This was discovered during purification and characterization of the Na⁺-NQR enzyme from Vibrio harveyi . The identification of this cofactor was tentative in earlier studies but has been confirmed in subsequent research.

To identify the flavin cofactor, researchers typically use techniques such as:

• UV-visible spectroscopy (characteristic absorption spectra)

• Fluorescence spectroscopy

• Mass spectrometry to confirm covalent attachment

• Biochemical assays to determine flavin type (FAD vs. FMN)

The covalent attachment of FMN to NqrC is a distinctive feature that differentiates it from some other flavoproteins where the flavin cofactor is non-covalently bound.

How conserved is the NqrC subunit sequence across different bacterial species?

Sequence alignment studies show that NqrC subunits are well conserved across various bacterial species. Analysis of NqrC from Vibrio harveyi, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Neisseria meningitidis, and Pseudomonas aeruginosa reveals significant homology .

Additionally, NqrC shows homology to paralogous RnfG subunits of the RNF complex from Escherichia coli and Vibrio cholerae . This conservation suggests the functional importance of this subunit across diverse bacterial species.

The conservation pattern can be visualized in the following representation based on sequence alignment data:

What are the most effective methods for expressing and purifying recombinant NqrC?

For successful expression and purification of recombinant NqrC from Vibrio harveyi, researchers should consider the following methodological approach:

• Expression system selection:

  • E. coli BL21(DE3) or similar strains are commonly used

  • Consider using pET expression vectors with T7 promoter for high-level expression

• Optimization of expression conditions:

  • Temperature: Lower temperatures (16-25°C) often improve protein folding

  • Induction: IPTG concentration between 0.1-0.5 mM

  • Duration: Extended expression times (16-24 hours) at lower temperatures

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Secondary purification: Ion exchange chromatography

  • Final polishing: Size exclusion chromatography

• Cofactor incorporation considerations:

  • Supplementation with FMN during expression may improve cofactor incorporation

  • Alternatively, reconstitution of the apo-protein with FMN post-purification

It's important to note that binding of free FMN to the apo-form of NqrC from Vibrio harveyi shows very low affinity in the absence of its partner proteins . Therefore, co-expression with other subunits or reconstitution strategies may be necessary for obtaining functionally active protein.

How can researchers effectively study the flavin binding properties of NqrC?

To study flavin binding properties of NqrC effectively, researchers should employ a multi-technique approach:

• Spectroscopic methods:

  • UV-visible spectroscopy to monitor flavin binding (absorbance at 450 nm)

  • Fluorescence spectroscopy (excitation at 450 nm, emission at 525 nm)

  • Circular dichroism to assess structural changes upon flavin binding

• Binding affinity determination:

  • Isothermal titration calorimetry (ITC)

  • Surface plasmon resonance (SPR)

  • Fluorescence quenching titrations

• Structural analysis of flavin binding site:

  • Site-directed mutagenesis of putative flavin-binding residues

  • X-ray crystallography or cryo-EM of NqrC with bound flavin

  • Molecular dynamics simulations to understand binding dynamics

• Analysis of covalent attachment:

  • Mass spectrometry to identify the specific residue involved in covalent attachment

  • Peptide mapping after limited proteolysis

  • Comparative analysis with other flavoproteins

Since NqrC shows very low affinity to FMN in the absence of its partner proteins , researchers should consider studying the protein in the context of the complete Na⁺-NQR complex or at least with its immediate interaction partners to obtain physiologically relevant results.

What functional assays can be used to characterize NqrC activity?

Several functional assays can be employed to characterize the activity of NqrC within the Na⁺-NQR complex:

• Electron transfer assays:

  • NADH:quinone oxidoreductase activity measurement using different quinone analogs

  • Spectrophotometric monitoring of NADH oxidation (decrease in absorbance at 340 nm)

  • Quinone reduction monitoring (species-dependent wavelengths)

• Na⁺ transport assays:

  • Reconstitution of purified Na⁺-NQR into proteoliposomes

  • Measurement of Na⁺ uptake using radioactive ²²Na⁺ or Na⁺-sensitive fluorescent dyes

  • Monitoring of membrane potential generation using voltage-sensitive dyes

• Specific NqrC function assessment:

  • Electron transfer from/to purified NqrC using artificial electron donors/acceptors

  • Redox titrations to determine the midpoint potential of the FMN cofactor

  • Stopped-flow kinetic measurements of electron transfer rates

• Inhibitor studies:

  • Characterization using specific Na⁺-NQR inhibitors like korormicin A

  • Competition assays with ubiquinone analogs

  • Structure-activity relationship analysis

When reconstituted into proteoliposomes, the Na⁺-NQR complex generates a transmembrane electric potential upon NADH:Q₁ oxidoreduction that is strictly dependent on Na⁺, resistant to protonophores like CCCP, and sensitive to sodium ionophores like ETH-157, confirming its function as a primary electrogenic sodium pump .

How do cryo-EM studies enhance our understanding of NqrC compared to earlier crystallographic approaches?

Recent cryo-EM studies have revolutionized our understanding of NqrC within the Na⁺-NQR complex in several key ways:

• Improved resolution of the entire complex:

  • Cryo-EM structures of Na⁺-NQR have been resolved at 2.5-3.1 Å resolution

  • This allows visualization of all six redox cofactors in their native arrangement

• More accurate cofactor distances:

  • Crystallographic structures suggested distances between redox cofactors (e.g., FMN and riboflavin) that were too long (29-32 Å edge-to-edge) for physiologically relevant electron transfer

  • Cryo-EM structures reveal that FMN in NqrC is positioned closer to other flavin cofactors (FMN in NqrB and riboflavin in NqrB) than previously thought

• Conformational flexibility insights:

  • Cryo-EM can capture different conformational states of the complex

  • Some regions show high flexibility, which may be functionally relevant for long-distance electron transfer

• Inhibitor binding sites:

  • Cryo-EM structures with bound inhibitors (korormicin A and aurachin D-42) reveal previously unresolved regions

  • The N-terminal region of NqrB, which is disordered in the absence of inhibitors, becomes ordered upon inhibitor binding

What is the arrangement of redox cofactors in NqrC relative to other subunits, and what are the implications for electron transfer?

The arrangement of redox cofactors in Na⁺-NQR, including the FMN cofactor in NqrC, creates an electron transfer pathway through the complex. Cryo-EM studies have revealed the following key features:

• Cofactor arrangement and distances:

  • The FMN cofactor in NqrC is positioned to facilitate electron transfer from/to cofactors in adjacent subunits

  • Edge-to-edge distances between cofactors are within the range required for physiologically relevant electron transfer (typically <14 Å)

• Proposed electron transfer pathway:

  • Electrons from NADH enter through FAD in NqrF

  • Transfer occurs through a series of redox cofactors, including the 2Fe-2S cluster and flavins

  • The FMN in NqrC serves as an intermediate electron carrier in this pathway

• Conformational changes during catalysis:

  • The arrangement suggests that conformational changes may be necessary to optimize electron transfer distances

  • The high flexibility of certain regions (e.g., parts of NqrF) may facilitate these conformational changes

• Implications for mechanism:

  • The positioning of NqrC's FMN relative to Na⁺-binding sites suggests its involvement in coupling electron transfer to Na⁺ translocation

  • The electron transfer through NqrC likely contributes to the generation of the electrochemical Na⁺ potential

This sophisticated arrangement of redox cofactors enables the complex to function as an efficient electrogenic sodium pump, converting the energy from NADH oxidation into a sodium gradient across the membrane.

What structural features of NqrC are involved in Na⁺ translocation?

While the exact mechanism of Na⁺ translocation by Na⁺-NQR remains under investigation, several structural features of NqrC are likely involved in this process:

• Transmembrane domain architecture:

  • NqrC contains transmembrane helices that contribute to forming the ion translocation pathway

  • The arrangement of these helices creates a conducive environment for Na⁺ binding and movement

• Charged residues:

  • Conserved negatively charged amino acids (Asp, Glu) within or adjacent to transmembrane segments may participate in Na⁺ coordination

  • These residues likely undergo conformational changes coupled to the redox state of cofactors

• Conformational coupling:

  • The covalently attached FMN in NqrC undergoes redox changes during catalysis

  • These redox changes likely trigger conformational alterations in the protein structure

  • Such conformational changes may alter the affinity for Na⁺ at specific binding sites, facilitating directional ion movement

• Interface with other subunits:

  • NqrC interacts with other subunits (particularly NqrB and NqrD) to form the complete Na⁺ translocation pathway

  • These interfaces are critical for maintaining the integrity of the ion conduction channel

Biochemical studies have shown that the Na⁺-NQR complex operates as a primary electrogenic sodium pump, with protons for ubiquinol formation being taken from the bacterial cytoplasm . The coordination between electron transfer through the redox cofactors (including NqrC's FMN) and Na⁺ movement through the transmembrane domains is essential for this energy-transducing function.

What evolutionary relationships exist between NqrC and similar proteins in other bacterial species?

NqrC exhibits notable evolutionary relationships with similar proteins across different bacterial species:

• Conservation among Na⁺-NQR-containing bacteria:

  • NqrC sequences show significant homology across various bacteria possessing the Na⁺-NQR complex

  • Sequence alignment reveals conservation patterns in Vibrio harveyi, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Neisseria meningitidis, and Pseudomonas aeruginosa

• Homology with RNF complex components:

  • NqrC shows homology to the RnfG subunits of the RNF complex from Escherichia coli and Vibrio cholerae

  • This suggests a potential evolutionary relationship between the Na⁺-NQR and RNF complexes

• Distribution across bacterial phyla:

  • Na⁺-NQR operons, including nqrC, are present in various bacterial lineages

  • Many pathogenic species possess this complex, suggesting its importance for bacterial adaptation

• Conservation of key functional domains:

  • Flavin-binding domains are highly conserved across species

  • Transmembrane segments show greater variability, potentially reflecting adaptation to specific ecological niches

The evolutionary conservation of NqrC underscores its functional importance in bacterial bioenergetics. The presence of Na⁺-NQR in numerous pathogenic bacteria also suggests its potential as a target for antimicrobial development.

How do environmental conditions affect the expression and function of NqrC in Vibrio harveyi?

Environmental conditions can significantly influence the expression and function of NqrC as part of the Na⁺-NQR complex in Vibrio harveyi:

• Salt concentration effects:

  • As a marine bacterium, V. harveyi typically grows in environments with elevated Na⁺ concentrations

  • The expression of the Na⁺-NQR complex, including NqrC, may be regulated in response to external Na⁺ levels

  • The function of Na⁺-NQR as a primary sodium pump is directly dependent on Na⁺ availability

• Stress response and gene expression:

  • Environmental stressors can trigger significant changes in V. harveyi gene expression

  • Stress conditions such as temperature shifts (37-46°C), ethanol exposure (4-16%), detergent exposure (0.14-0.56 mM SDS), and pH changes (0.04-0.05 M NaOH, 0.012-0.024 M HCl) have been shown to affect gene expression patterns in V. harveyi

  • These stress responses may indirectly affect nqrC expression

• Quorum sensing and NqrC regulation:

  • V. harveyi utilizes sophisticated quorum sensing systems involving multiple autoinducers

  • While direct regulation of nqrC by quorum sensing has not been explicitly demonstrated, quorum sensing controls numerous cellular processes in V. harveyi

  • The energy metabolism (which involves Na⁺-NQR) may be coordinated with population density through quorum sensing mechanisms

• Oxygen availability:

  • As Na⁺-NQR functions in aerobic respiration, oxygen availability influences its activity

  • Transition between aerobic and anaerobic growth conditions likely affects the expression and function of the complex

Understanding how environmental conditions affect NqrC expression and function could provide insights into the adaptation of V. harveyi to various ecological niches and stress conditions.

What are the main challenges in expressing functional recombinant NqrC and how can they be overcome?

Researchers face several challenges when expressing functional recombinant NqrC, with corresponding solutions:

• Cofactor incorporation difficulties:

  • Challenge: NqrC requires covalently attached FMN, which may not be efficiently incorporated during heterologous expression

  • Solution:

    • Supplement expression media with riboflavin/FMN

    • Co-express with enzymes involved in flavin attachment

    • Develop in vitro flavinylation protocols

• Low protein solubility:

  • Challenge: As a membrane-associated protein, NqrC may have solubility issues

  • Solution:

    • Use solubility-enhancing fusion tags (MBP, SUMO, etc.)

    • Optimize expression temperature (typically lower temperatures improve solubility)

    • Screen different detergents for extraction and purification

• Protein instability without partner subunits:

  • Challenge: NqrC shows very low affinity to FMN in the absence of partner proteins

  • Solution:

    • Co-express with other Na⁺-NQR subunits

    • Design constructs that include stabilizing domains from partner proteins

    • Develop reconstitution protocols with other purified subunits

• Proper membrane integration:

  • Challenge: Ensuring correct topology and membrane insertion

  • Solution:

    • Use specialized expression systems designed for membrane proteins

    • Consider cell-free expression systems with supplied lipids/nanodiscs

    • Employ fluorescence-based assays to confirm proper membrane insertion

• Functional verification difficulties:

  • Challenge: Assessing functionality of isolated NqrC outside the complete complex

  • Solution:

    • Develop specific activity assays focused on NqrC's partial reactions

    • Use spectroscopic methods to verify correct cofactor incorporation and environment

    • Employ biophysical techniques to confirm proper folding and stability

By addressing these challenges systematically, researchers can improve the yield and quality of functional recombinant NqrC for structural and biochemical studies.

What advanced spectroscopic techniques are most informative for studying NqrC redox properties?

Several advanced spectroscopic techniques provide valuable insights into the redox properties of NqrC:

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Application: Detects unpaired electrons in semiquinone radical intermediates of FMN

  • Information provided: Identifies radical species, determines g-values characteristic of the flavin environment

  • Advantage: Can detect transient radical species during electron transfer

• Resonance Raman Spectroscopy:

  • Application: Probes the vibrational modes of the flavin cofactor

  • Information provided: Details about flavin-protein interactions and changes during redox transitions

  • Advantage: Can be performed with relatively small amounts of protein

• Time-resolved fluorescence spectroscopy:

  • Application: Monitors changes in flavin fluorescence during redox reactions

  • Information provided: Kinetic parameters of electron transfer reactions involving NqrC

  • Advantage: Can track reactions on nanosecond to millisecond timescales

• Protein Film Voltammetry:

  • Application: Direct electrochemical measurement of NqrC redox properties

  • Information provided: Precise determination of redox potentials and electron transfer rates

  • Advantage: Allows control of experimental conditions (pH, temperature, etc.)

• FTIR Difference Spectroscopy:

  • Application: Detects structural changes coupled to redox transitions

  • Information provided: Identifies specific amino acid residues involved in redox-linked conformational changes

  • Advantage: High sensitivity to subtle structural alterations

Each technique provides complementary information, and a multi-technique approach is typically most informative for thoroughly characterizing the redox properties of NqrC and understanding its role in the electron transfer pathway of Na⁺-NQR.

How can researchers effectively study the interaction between NqrC and other subunits of the Na⁺-NQR complex?

To effectively study interactions between NqrC and other Na⁺-NQR subunits, researchers can employ various complementary approaches:

• Co-purification and pull-down assays:

  • Tag one subunit (e.g., His-tagged NqrC) and identify co-purifying partners

  • Verify specific interactions using controls and quantitative binding measurements

  • Map interaction domains by testing truncated constructs

• Crosslinking coupled with mass spectrometry:

  • Use chemical crosslinkers to capture transient protein-protein interactions

  • Identify crosslinked peptides by mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Förster Resonance Energy Transfer (FRET):

  • Label NqrC and potential partner subunits with suitable fluorophore pairs

  • Measure energy transfer as indication of proximity

  • Determine distance constraints between subunits

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize purified NqrC and measure binding kinetics with other subunits

  • Determine association and dissociation rates, and binding affinities

  • Test effects of mutations or ligands on binding properties

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Compare deuterium uptake patterns of individual subunits versus assembled complex

  • Identify regions protected from exchange due to protein-protein interactions

  • Map conformational changes induced by complex formation

• Cryo-EM of sub-complexes:

  • Generate and purify defined sub-complexes containing NqrC

  • Determine structures at various assembly stages

  • Identify conformational changes associated with complex assembly

• Genetic approaches:

  • Create site-directed mutants targeting predicted interface residues

  • Assess impact on complex assembly and function

  • Perform suppressor mutation analysis to identify compensatory changes

By combining these approaches, researchers can build a comprehensive understanding of how NqrC interacts with other subunits within the Na⁺-NQR complex, which is essential for understanding the mechanism of coupled electron transfer and Na⁺ translocation.

How might understanding NqrC function contribute to developing new antimicrobial strategies?

Understanding NqrC function could contribute to novel antimicrobial strategies in several ways:

• Na⁺-NQR as a pathogen-specific target:

  • Na⁺-NQR is present in many pathogenic bacteria including Vibrio species, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Neisseria meningitidis, and Pseudomonas aeruginosa

  • The complex is absent in mammals, making it an attractive target for selective inhibition

  • Targeting NqrC could disrupt bacterial bioenergetics without affecting host metabolism

• Inhibitor development:

  • Structure-guided design of inhibitors targeting NqrC's flavin binding site

  • Compounds that interfere with NqrC's interaction with other subunits

  • Molecules that block electron transfer through the NqrC cofactor

• Exploitation of species-specific differences:

  • Comparative analysis of NqrC across pathogenic species could reveal unique structural features

  • These differences could be exploited to develop species-selective antimicrobials

  • Such selectivity could help preserve beneficial microbiota while targeting specific pathogens

• Combination therapy approaches:

  • Na⁺-NQR inhibitors targeting NqrC could sensitize bacteria to existing antibiotics

  • Disruption of bioenergetics may impair efflux pump function, reducing antibiotic resistance

  • Synergistic effects could allow lower doses of conventional antibiotics

• Impact on virulence and persistence:

  • Interference with Na⁺-NQR function may affect bacterial adaptation to host environments

  • Studies on V. harveyi show that environmental stresses affect bacterial gene transfer capabilities

  • Targeting NqrC might reduce bacterial persistence under stress conditions

The development of specific inhibitors targeting the Na⁺-NQR complex, potentially through the NqrC subunit, represents a promising approach for addressing the growing challenge of antibiotic resistance in pathogenic bacteria.

What are the most pressing unresolved questions regarding NqrC structure and function?

Despite significant advances, several critical questions about NqrC remain unresolved:

• Precise mechanism of covalent flavin attachment:

  • Which specific residue(s) form the covalent bond with FMN?

  • What enzymes or cofactors are required for the flavinylation process?

  • How is this process regulated during protein biogenesis?

• Detailed electron transfer pathway:

  • What are the exact kinetic parameters for electron transfer to/from NqrC?

  • How do conformational changes modulate electron transfer rates?

  • What is the precise sequence of electron movement through the complex?

• Coupling mechanism to Na⁺ translocation:

  • How are redox changes in NqrC's FMN coupled to Na⁺ movement?

  • Which specific residues in NqrC participate in Na⁺ binding or channel formation?

  • What conformational changes link electron transfer to ion translocation?

• Regulatory mechanisms:

  • How is NqrC expression regulated in response to environmental conditions?

  • Are there post-translational modifications that affect NqrC function?

  • How is the assembly of NqrC into the complex coordinated and regulated?

• Species-specific adaptations:

  • How do variations in NqrC sequence across species reflect adaptation to different ecological niches?

  • Do these adaptations affect substrate specificity, inhibitor sensitivity, or ion selectivity?

  • What evolutionary pressures have shaped NqrC diversification?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, biophysics, and molecular genetics. Resolving these issues will provide a more complete understanding of NqrC's role in bacterial bioenergetics and potentially reveal new targets for antimicrobial development.

What emerging technologies are advancing research on NqrC and the Na⁺-NQR complex?

Several cutting-edge technologies are driving advances in NqrC and Na⁺-NQR research:

• Advanced cryo-EM methodologies:

  • Time-resolved cryo-EM to capture different functional states

  • Improved resolution enabling visualization of water molecules and ions

  • Computational classification approaches to identify multiple conformational states

  • These advances have already improved our understanding of Na⁺-NQR structure beyond what was possible with crystallography

• Integrative structural biology approaches:

  • Combining cryo-EM with mass spectrometry, EPR, and computational modeling

  • Cross-linking mass spectrometry to map protein-protein interactions

  • Small-angle X-ray scattering (SAXS) to study conformational dynamics

  • These integrated approaches provide a more complete picture of complex structure and dynamics

• Advanced spectroscopic techniques:

  • Ultra-fast spectroscopy to track electron transfer in real-time

  • Single-molecule spectroscopy to observe heterogeneity in electron transfer events

  • 2D IR spectroscopy to detect subtle conformational changes coupled to catalysis

• Genetic tools for bacterial systems:

  • CRISPR-Cas9 genome editing in bacterial species containing Na⁺-NQR

  • Improved methods for generating site-directed mutations in Vibrio species

  • High-throughput phenotypic screening approaches

  • Recent advances in V. harveyi genetic manipulation through stress-induced conjugation techniques

• Computational approaches:

  • Molecular dynamics simulations of the entire Na⁺-NQR complex

  • Quantum mechanical/molecular mechanical (QM/MM) calculations to study electron transfer

  • Machine learning approaches to predict protein-protein interactions and drug binding

• Synthetic biology and protein engineering:

  • Designer Na⁺-NQR complexes with modified subunits for specific functions

  • Biosensor development based on Na⁺-NQR components

  • Directed evolution approaches to enhance desired properties

These technological advances are enabling researchers to address long-standing questions about NqrC function and providing new opportunities for applications in biotechnology and medicine.