Recombinant Vibrio vulnificus Na (+)-translocating NADH-quinone reductase subunit F (nqrF), partial

What is the biochemical role of the nqrF subunit within the NQR complex of Vibrio vulnificus?

The nqrF subunit serves as the peripheral component of the Na⁺-translocating NADH:quinone oxidoreductase (NQR) complex and performs the critical function of NADH oxidation. This subunit hosts one flavin adenine dinucleotide (FAD) and a 2Fe-2S cluster as cofactors, making it essential for the initial electron transfer steps in the respiratory chain of V. vulnificus . By catalyzing the oxidation of NADH, nqrF initiates the electron transfer pathway that ultimately couples with Na⁺ translocation across the bacterial membrane, generating the sodium motive force (SMF) that drives various cellular processes including flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport .

How does the nqrF subunit interact with other components of the NQR complex?

The nqrF subunit functions within a six-subunit membrane protein complex encoded by the consecutive structural genes nqrABCDEF. As the peripheral component, nqrF interfaces with the membrane-bound subunits while maintaining its NADH oxidation capability. Electrons transferred from NADH oxidation at nqrF are subsequently passed to other redox centers within the complex, including the covalently attached FMN molecules in subunits NqrB and NqrC, and the Fe center ligated by membrane-bound subunits NqrD and NqrE . This electron transfer pathway ultimately reduces ubiquinone while coupling with Na⁺ translocation. The maturation of this complex also involves coordination with the ApbE protein (encoded by the gene immediately downstream of nqrF), which functions as a flavin insertase necessary for the proper attachment of FMN to NqrB and NqrC subunits .

What are the key structural features of recombinant nqrF that impact its functionality?

Recombinant nqrF's functionality depends on several critical structural elements:

• Redox Centers: The subunit contains one FAD molecule and a 2Fe-2S cluster, both serving as essential redox cofactors . These centers must be properly incorporated during protein expression and purification to maintain enzymatic activity.

• NADH Binding Domain: nqrF possesses a specific binding pocket for NADH that positions the substrate optimally for electron transfer to the FAD cofactor.

• Interaction Surfaces: Specific amino acid sequences and structural motifs facilitate proper interaction with other NQR subunits, particularly those required for electron transfer from nqrF to subsequent components in the chain.

• Membrane Association Elements: While nqrF is considered a peripheral subunit, it must maintain appropriate association with the membrane components of the NQR complex.

These structural features collectively enable nqrF to perform its primary function of NADH oxidation and subsequent electron transfer within the respiratory chain of V. vulnificus.

How does the Fe-S cluster in nqrF affect electron transfer efficiency and what factors influence its assembly?

The 2Fe-2S cluster in nqrF serves as a critical intermediate in the electron transport pathway. The proper assembly and maintenance of this cluster significantly impacts the electron transfer efficiency of the entire NQR complex. Several factors influence Fe-S cluster assembly in nqrF:

• Iron Availability: The NqrM protein, encoded by the nqrM gene, is presumed to function as an Fe delivery protein essential for proper Fe-S cluster formation .

• Redox Environment: The oxidation state of the environment affects Fe-S cluster stability and assembly. Reducing conditions may destabilize certain Fe-S configurations.

• Protein Folding: Proper tertiary structure formation is necessary to create the appropriate ligand environment for the Fe-S cluster.

• Accessory Proteins: Assembly of the Fe-S cluster likely requires specific Fe-S biogenesis machinery proteins that aren't fully characterized in the search results but would be consistent with typical bacterial Fe-S cluster formation.

What are the optimal expression systems and conditions for producing functional recombinant nqrF?

Expression Systems and Optimized Conditions:

• Host Selection: E. coli expression systems (particularly BL21(DE3) or derivatives) are commonly used for recombinant nqrF expression. These systems can be engineered to co-express iron-sulfur cluster assembly machinery if needed.

• Vector Design: Expression vectors should include appropriate promoter systems (T7 or similar inducible promoters), affinity tags for purification (His-tag preferable), and consider codon optimization for the host organism.

• Growth Conditions:

  • Temperature: Lower induction temperatures (16-18°C) often improve folding and solubility

  • Media: Iron-supplemented media enhances Fe-S cluster incorporation

  • Induction: Gradual induction with lower IPTG concentrations (0.1-0.5 mM)

  • Aeration: Proper aeration is crucial for Fe-S protein expression

• Co-expression Strategies: Co-expression with nqrM (the presumed Fe delivery protein) may enhance proper Fe-S cluster formation . Additionally, co-expression with specific iron-sulfur cluster assembly proteins could improve yield of functional protein.

• Extraction Conditions: Careful cell lysis under mildly reducing conditions helps preserve the integrity of the Fe-S clusters while avoiding excessive reduction that might destabilize these structures.

What analytical methods are most effective for evaluating nqrF activity and integrity?

Analytical Methods for nqrF Characterization:

• Spectroscopic Analyses:

  • UV-Visible spectroscopy: Characteristic absorption patterns for FAD and Fe-S clusters

  • Electron Paramagnetic Resonance (EPR): Evaluation of Fe-S cluster integrity and oxidation state

  • Circular Dichroism (CD): Secondary structure analysis

• Functional Assays:

  • NADH oxidation activity: Spectrophotometric monitoring of NADH consumption at 340 nm

  • Electron transfer ability: Using artificial electron acceptors like ferricyanide

  • Ubiquinone reduction: When testing in context of complete NQR complex

• Structural Analysis:

  • Size exclusion chromatography: Assessment of oligomeric state and complex formation

  • Native PAGE: Evaluation of protein integrity and complex assembly

  • Thermal shift assays: Stability assessment under various conditions

• Advanced Techniques:

  • Protein film voltammetry: Direct measurement of electron transfer capabilities

  • Reconstitution studies: Testing functionality in artificial membrane systems

  • AlphaFold2 modeling: As recently employed for related Vibrio proteins to predict structures and interactions

These methods collectively provide comprehensive assessment of both the structural integrity and functional capacity of recombinant nqrF.

How can recombinant nqrF be utilized to study energy transduction mechanisms in Vibrio species?

Recombinant nqrF provides a valuable tool for investigating the mechanistic details of energy transduction in Vibrio species through several advanced approaches:

• Mutagenesis Studies: Site-directed mutagenesis of key residues in nqrF can elucidate critical amino acids involved in NADH binding, FAD coordination, Fe-S cluster ligation, or interaction with other NQR subunits. These studies help map the electron transfer pathway and identify rate-limiting steps in the energy transduction process.

• Reconstitution Experiments: Purified recombinant nqrF can be incorporated into liposomes or nanodiscs containing other NQR components to study the minimal requirements for Na⁺ translocation and establish structure-function relationships.

• Comparative Analyses: Recombinant nqrF from various Vibrio species can be compared to identify adaptations in energy transduction mechanisms. This approach is particularly valuable considering the pathogenic nature of V. vulnificus versus other Vibrio species.

• Inhibitor Development: As NQR is the main producer of the sodium motive force that drives numerous cellular processes , recombinant nqrF can serve as a target for inhibitor screening to develop potential antimicrobial compounds against Vibrio species.

• Inter-System Compatibility: The ability of nqrF to interact with components from other bacterial respiratory systems can be assessed, providing insights into the evolution and adaptation of energy transduction mechanisms.

These applications contribute to our fundamental understanding of bacterial bioenergetics while potentially identifying novel targets for antibacterial therapy.

What role does nqrF play in iron homeostasis and respiratory adaptation in Vibrio vulnificus?

The NQR complex, including its nqrF subunit, appears to have significant implications for iron homeostasis and respiratory adaptation in V. vulnificus:

• Iron Metabolism Interconnection: Quantitative RT-PCR studies have revealed that NQR mutations influence iron uptake and homeostasis gene expression in V. cholerae, suggesting similar mechanisms may exist in V. vulnificus . The nqrF subunit, with its Fe-S cluster, represents a direct link between respiratory function and iron utilization.

• Adaptive Responses: Under iron-limited conditions (such as those encountered during host infection), the expression and activity of nqrF may be modulated to optimize respiratory efficiency while maintaining iron homeostasis.

• Growth Environment Adaptation: The NQR complex's role as the main producer of sodium motive force suggests that nqrF activity may adapt to different growth environments, including varying sodium concentrations encountered in estuarine habitats and human hosts.

• Regulatory Networks: The connection between NQR and iron metabolism suggests nqrF may be regulated as part of global stress responses that coordinate respiratory function with nutrient availability.

• Virulence Connection: While not directly established for nqrF, the NQR complex's influence on energy production may indirectly affect virulence factor expression and function in V. vulnificus, particularly considering the pathogen's ability to cause severe infections and its production of toxins like MARTX .

Understanding these relationships provides insights into how V. vulnificus adapts to diverse environments and maintains metabolic flexibility during infection processes.

What are common pitfalls in purification and characterization of recombinant nqrF and how can they be addressed?

Common Challenges and Solutions:

Additional considerations include maintaining appropriate redox conditions throughout purification to preserve Fe-S cluster integrity and employing spectroscopic techniques to monitor cofactor status at each purification stage.

How can researchers address data inconsistencies when comparing recombinant nqrF function with native NQR complex activity?

When comparing recombinant nqrF function with native NQR complex activity, researchers frequently encounter data inconsistencies that require methodological solutions:

• Context-Dependent Activity Variations: Recombinant nqrF in isolation often shows different kinetic parameters compared to its behavior within the native complex. To address this:

  • Develop reconstitution systems that incorporate other NQR components

  • Establish normalization factors based on well-characterized activities

  • Use partial complexes (e.g., nqrF with nqrC) as intermediate comparison points

• Post-Translational Modification Differences: Native nqrF may contain modifications absent in recombinant systems:

  • Analyze native protein by mass spectrometry to identify modifications

  • Engineer expression systems to incorporate relevant modifications

  • Document modification-dependent activity differences

• Cofactor Heterogeneity: Variations in Fe-S cluster and FAD incorporation lead to heterogeneous preparations:

  • Develop spectroscopic methods to quantify cofactor incorporation ratios

  • Normalize activity data to cofactor content rather than protein concentration

  • Establish reconstitution protocols for depleted cofactors

• Membrane Environment Effects: The native membrane environment significantly impacts NQR function:

  • Utilize nanodiscs or liposomes with defined lipid compositions

  • Compare detergent-solubilized native complex with recombinant proteins in identical detergent systems

  • Develop membrane-mimetic systems that better replicate the native environment

• Interspecies Variations: When comparing nqrF from different Vibrio species:

  • Account for sequence variations through structural modeling

  • Establish standardized activity assays applicable across species

  • Document species-specific cofactor preferences or kinetic parameters

Careful documentation of these variables and standardization of experimental conditions will improve data consistency and interpretability across different research groups.

How might recombinant nqrF contribute to understanding pathogenicity mechanisms in Vibrio vulnificus?

Recombinant nqrF represents a valuable tool for investigating the relationship between respiratory energy metabolism and virulence in V. vulnificus through several research avenues:

• Energy-Virulence Coupling: The NQR complex, with nqrF as a key component, generates the sodium motive force that powers numerous cellular processes . Research using recombinant nqrF variants could establish how changes in respiratory efficiency affect the expression and activity of known virulence factors, including the MARTX toxin identified as crucial for V. vulnificus pathogenicity .

• Host Environment Adaptation: V. vulnificus encounters varying sodium concentrations and iron availability within host environments. Studies employing recombinant nqrF under simulated host conditions could reveal how respiratory adaptation contributes to survival during infection.

• Toxin Production Correlation: Recent research has shown that V. vulnificus produces the MARTX toxin with different arrangements of effector domains . Investigating whether NQR activity, particularly nqrF function, correlates with specific toxin variant expression could provide insights into virulence regulation.

• Inhibitor Development: As a key component of energy metabolism, nqrF represents a potential target for antimicrobial development. Structural and functional studies of recombinant nqrF could facilitate the design of inhibitors that specifically target pathogenic Vibrio species.

• Protein-Protein Interaction Studies: The recent discovery that MCF toxin (within MARTX) interacts with human proteins like ARFs and Rab family GTPases raises the question of whether respiratory components like nqrF might also interact with host factors during infection.

These research directions could significantly enhance our understanding of how respiratory metabolism contributes to the remarkable virulence of V. vulnificus, which is associated with 1% of all food-related deaths despite its relatively low incidence .

What computational approaches can enhance structural and functional predictions for nqrF variants?

Advanced Computational Approaches for nqrF Research:

• AlphaFold2 and Related Structural Prediction Tools:

  • The successful application of AlphaFold2 to predict structures of MCF toxin interactions with human proteins demonstrates the value of these approaches for Vibrio proteins

  • Structural predictions for nqrF variants can identify critical residues involved in cofactor binding and protein-protein interactions

  • Integration of experimental data with computational models can refine predictions and guide experimental design

• Molecular Dynamics Simulations:

  • Simulations of nqrF in different redox states can reveal conformational changes during electron transfer

  • Integration of Fe-S cluster parameters into simulation frameworks allows dynamic modeling of electron transfer pathways

  • Membrane-protein interaction simulations can elucidate how nqrF associates with other NQR components

• Quantum Mechanical/Molecular Mechanical (QM/MM) Approaches:

  • Hybrid methods can specifically model electron transfer events between NADH, FAD, and the Fe-S cluster

  • Energy barrier calculations can identify rate-limiting steps in the reaction mechanism

  • Redox potential predictions can guide experimental validation

• Systems Biology Integration:

  • Network modeling connecting nqrF activity to broader metabolic and virulence networks

  • Flux balance analysis incorporating NQR activity parameters

  • Multi-scale models linking molecular function to cellular phenotypes

• Comparative Genomics and Evolutionary Analysis:

  • Analysis of nqrF sequence variation across Vibrio species can identify conserved functional motifs

  • Correlation of sequence variations with pathogenicity profiles may reveal virulence-associated adaptations

  • Evolutionary trajectory reconstruction to understand the development of specialized functions

These computational approaches complement experimental methods and can significantly accelerate research by generating testable hypotheses about nqrF structure-function relationships.

What experimental design is optimal for investigating the interaction between nqrF and nqrM in Fe-S cluster biogenesis?

To effectively investigate the interaction between nqrF and nqrM in Fe-S cluster biogenesis, a multi-faceted experimental approach is recommended:

• Co-expression and Co-purification Studies:

  • Design constructs for co-expression of tagged nqrF and nqrM

  • Implement tandem affinity purification to isolate the complex

  • Use size exclusion chromatography to evaluate complex formation

  • Compare Fe-S cluster incorporation efficiency in co-expression versus individual expression systems

• Protein-Protein Interaction Validation:

  • Apply microscale thermophoresis or isothermal titration calorimetry to quantify binding affinities

  • Conduct pull-down assays with immobilized nqrM to capture nqrF

  • Perform crosslinking mass spectrometry to identify interaction interfaces

  • Use FRET pairs for real-time monitoring of interactions in vitro

• Fe-S Cluster Transfer Assays:

  • Develop spectroscopic methods to monitor Fe-S cluster transfer from nqrM to apo-nqrF

  • Implement time-resolved measurements of cluster assembly

  • Assess the impact of different redox conditions on transfer efficiency

  • Compare transfer rates with known Fe-S carrier proteins

• Mutagenesis Studies:

  • Design mutations in potential Fe-binding residues of nqrM

  • Create corresponding mutations in potential cluster-binding residues of nqrF

  • Evaluate the impact of mutations on complex formation and cluster transfer

• In vivo Validation:

  • Develop fluorescent protein fusions to monitor localization and interaction

  • Implement bacterial two-hybrid systems to confirm interactions

  • Construct nqrM knockout/knockdown systems to assess impact on functional nqrF levels

  • Measure Fe-S enzyme activities as readouts for successful cluster biogenesis

This comprehensive approach addresses both the physical interaction between these proteins and the functional consequences for Fe-S cluster assembly and incorporation into nqrF.

How can researchers effectively measure the impact of nqrF mutations on sodium translocation and respiratory efficiency?

Methodological Framework for Evaluating nqrF Mutation Effects:

• Whole-Cell Bioenergetic Measurements:

  • Develop a library of V. vulnificus strains expressing nqrF variants

  • Measure sodium-dependent respiration rates using oxygen electrodes

  • Quantify membrane potential using voltage-sensitive fluorescent dyes

  • Compare growth rates in sodium-rich versus sodium-limited media

  • Assess sensitivity to sodium gradient disruptors

• Membrane Vesicle Studies:

  • Prepare inside-out membrane vesicles from cells expressing nqrF variants

  • Measure NADH-driven sodium uptake using sodium-sensitive fluorophores

  • Determine sodium:electron coupling ratios for different mutations

  • Assess the impact of sodium concentration on electron transfer rates

• Reconstituted Systems:

  • Purify NQR complexes containing nqrF variants

  • Reconstitute into proteoliposomes with controlled lipid composition

  • Measure sodium pumping activity using entrapped indicators or 22Na+ uptake

  • Correlate electron transfer rates with sodium translocation efficiency

• Structure-Function Analysis:

  • Target mutations to predicted sodium pathway residues

  • Create conservative versus non-conservative substitutions

  • Evaluate the impact on both electron transfer and sodium pumping

  • Correlate functional changes with structural predictions

• Physiological Impact Assessment:

  • Measure swimming behavior and flagellar activity in nqrF mutants

  • Assess ATP production efficiency

  • Evaluate survival under energy-limited conditions

  • Quantify expression of compensatory bioenergetic systems

Data Analysis and Validation:

This methodological framework provides comprehensive evaluation of how specific nqrF mutations affect both the direct biochemical activities of the NQR complex and their physiological consequences in V. vulnificus.