Recombinant Shewanella halifaxensis ATP synthase subunit c (atpE)

What is the function of ATP synthase subunit c (atpE) in Shewanella halifaxensis?

ATP synthase subunit c (atpE) in Shewanella halifaxensis forms part of the c-ring in the F₀ region of the ATP synthase complex. This subunit plays a crucial role in the transmembrane proton translocation process that drives ATP synthesis. Each c-subunit contains a conserved carboxylate residue (typically glutamate) that is essential for proton binding and transport across the membrane. During ATP synthesis, protons from the periplasm bind to this glutamate residue, allowing rotation of the c-ring which drives conformational changes in the F₁ region, ultimately leading to ATP production from ADP and inorganic phosphate .

The c-ring essentially functions as a rotor that converts the energy of the proton motive force into mechanical rotation. This rotation is then transmitted to the central stalk of the ATP synthase, leading to conformational changes in the catalytic sites located in the F₁ region where ATP synthesis occurs. The number of c-subunits in the ring can vary between species, with most bacterial ATP synthases containing 10-15 subunits .

How does ATP synthase subunit c contribute to the proton motive force in bacteria?

The c-ring of ATP synthase harnesses this pre-existing PMF through a two-half-channel mechanism. Protons enter through a periplasmic half-channel in subunit a, bind to the conserved glutamate residue (Glu 56 in some bacterial systems) of a c-subunit, and this protonation allows rotation of the c-ring. As the ring rotates, the protonated glutamate moves through the lipid bilayer until it reaches the cytoplasmic half-channel, where the proton is released due to interaction with a positively charged arginine in subunit a . Each complete rotation of the c-ring, driven by sequential protonation and deprotonation of multiple c-subunits, results in the synthesis of multiple ATP molecules.

What are the key structural features of bacterial ATP synthase subunit c?

Bacterial ATP synthase subunit c is a small, hydrophobic protein that typically consists of two transmembrane α-helices connected by a polar loop region exposed to the cytoplasm. The key structural features include:

• Conserved Carboxylate Residue: Each c-subunit contains a highly conserved carboxylate residue (usually glutamate, such as Glu 56 in some bacterial systems) located on the second transmembrane helix. This residue is essential for proton binding and translocation .

• Hairpin Structure: The two α-helices form a hairpin-like structure that spans the membrane, with both the N and C termini located on the cytoplasmic side of the membrane in most bacteria.

• Oligomeric Ring Formation: Multiple c-subunits associate to form a ring structure (c-ring) through hydrophobic interactions. The number of c-subunits in the ring can vary between species, typically ranging from 8 to 15 subunits .

• Interface with Subunit a: The outer surface of the c-ring interacts with subunit a to form the proton translocation pathway. This interface is critical for the function of the ATP synthase and contains the two half-channels for proton entry and exit .

The structure allows the c-ring to function as a rotor that converts the energy of proton flow down the electrochemical gradient into mechanical rotation, which drives ATP synthesis in the F₁ portion of the complex.

How can recombinant Shewanella halifaxensis ATP synthase subunit c be expressed in laboratory settings?

Expression of recombinant Shewanella halifaxensis ATP synthase subunit c (atpE) requires careful consideration of the protein's hydrophobic nature and membrane localization. A methodological approach includes:

• Vector Selection and Design:

  • Use expression vectors with strong, inducible promoters (T7, tac) compatible with E. coli.

  • Include affinity tags (His6, Strep-tag II) for purification, preferably at the C-terminus to minimize interference with membrane insertion.

  • Consider fusion partners (MBP, SUMO) to improve solubility and expression.

• Host Strain Selection:

  • E. coli C41(DE3) or C43(DE3) strains are often preferred for membrane protein expression.

  • BL21(DE3) derivatives with modifications to reduce proteolysis (e.g., lacking lon and ompT proteases) may improve yield.

• Expression Conditions:

  • Induce at lower temperatures (16-25°C) to slow protein synthesis and allow proper membrane insertion.

  • Use lower inducer concentrations to prevent inclusion body formation.

  • Supplement with specific lipids if necessary to stabilize the protein.

• Extraction and Purification:

  • Use gentle detergents (DDM, GDN) for membrane solubilization, similar to those used for Bacillus PS3 ATP synthase .

  • Apply affinity chromatography followed by size exclusion chromatography for purification.

  • Consider lipid supplementation during purification to maintain protein stability.

The method described for Bacillus PS3 ATP synthase purification provides a useful template, involving solubilization with glycol-diosgenin (GDN), affinity purification via a His-tag, and final purification using size exclusion chromatography .

What are the optimal conditions for expressing and purifying functional recombinant Shewanella halifaxensis ATP synthase subunit c?

Optimizing expression and purification of functional recombinant Shewanella halifaxensis ATP synthase subunit c requires careful consideration of multiple parameters:

Expression Optimization Table:

Purification Strategy:

• Membrane Isolation: Thoroughly wash membranes with high-salt buffer (300 mM NaCl) to remove peripherally associated proteins.

• Solubilization: Use mild detergents that preserve c-ring integrity. Based on successful approaches with other bacterial ATP synthases, glycol-diosgenin (GDN) at 1% (w/v) with gentle mixing at room temperature for 1 hour has proven effective . Alternative detergents include dodecyl maltoside (DDM) at 1% or digitonin at 2%.

• Affinity Purification: Implement a two-step affinity purification using a HisTrap column with 20 mM imidazole in the binding buffer and 200 mM imidazole for elution, as demonstrated for Bacillus PS3 ATP synthase .

• Size Exclusion Chromatography: Use a Superose 6 increase column equilibrated with buffer containing 20 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 10% glycerol, 150 mM NaCl, and 0.02% detergent .

• Stability Optimization: Include stabilizing agents such as 5 mM 6-aminocaproic acid and 5 mM benzamidine throughout the purification process to prevent proteolysis .

This methodological approach needs to be empirically tested and optimized specifically for Shewanella halifaxensis ATP synthase subunit c, as the optimal conditions may differ from those used for other bacterial ATP synthases.

How can site-directed mutagenesis be applied to study the function of specific residues in Shewanella halifaxensis ATP synthase subunit c?

Site-directed mutagenesis is a powerful approach to investigate the functional significance of specific residues in Shewanella halifaxensis ATP synthase subunit c. A comprehensive methodology includes:

Strategic Residue Selection:

• Conserved Proton-Binding Site: The conserved glutamate residue (equivalent to Glu 56 in some bacterial systems) is critical for proton binding and translocation . Mutations like E→Q, E→D, or E→A can reveal the importance of carboxyl group chemistry, side chain length, and protonation state.

• Helix-Helix Interface Residues: Residues at the interface between adjacent c-subunits are crucial for c-ring assembly and stability. Conservative substitutions (e.g., L→I, V→I) can reveal the tolerance for structural variations.

• Subunit-a Interface Residues: Residues that interact with subunit a are essential for forming the proton translocation pathway . Mutations at this interface can reveal the importance of specific interactions for proton transport.

Experimental Protocol:

• Mutagenesis Approach: Use overlap extension PCR or commercial kits (Q5 Site-Directed Mutagenesis Kit) to introduce specific mutations into the atpE gene.

• Expression and Purification: Express and purify mutant proteins using the conditions established for the wild-type protein, being mindful that some mutations may affect expression or stability.

• Functional Assays:

  • Proton Pumping Activity: Use a high-precision pH meter to monitor pH variations in cell suspensions expressing mutant proteins, similar to the approach used for studying proton pumping in recombinant S. oneidensis .

  • ATP Synthesis Assay: Measure ATP production in membrane vesicles or reconstituted proteoliposomes under conditions that generate a proton motive force.

  • ATP Hydrolysis Assay: Assess the reverse reaction (ATP hydrolysis coupled to proton pumping) to comprehensively understand the effect of mutations.

• Structural Analysis:

  • Utilize cryo-EM to determine the structure of mutant proteins and compare with wild-type to identify structural changes.

  • Apply molecular dynamics simulations to predict the effects of mutations on protein dynamics and proton transport.

Data Analysis Table for Mutational Studies:

This systematic approach allows researchers to establish structure-function relationships and understand the molecular mechanism of proton translocation and ATP synthesis in Shewanella halifaxensis ATP synthase.

What techniques are most effective for studying the rotational dynamics of the c-ring in Shewanella ATP synthase?

Studying the rotational dynamics of the ATP synthase c-ring requires specialized techniques that can capture the nanoscale movements of this complex molecular machine:

1. Single-Molecule Fluorescence Resonance Energy Transfer (smFRET):

• Methodology: Attach donor and acceptor fluorophores to specific sites on the c-ring and stationary parts of the ATP synthase (e.g., subunit a).

• Analysis: Monitor changes in FRET efficiency corresponding to rotational motion with millisecond time resolution.

• Advantages: Provides information about rotational step size, dwell times, and heterogeneity in motion.

• Implementation: Requires careful selection of labeling sites to minimize functional interference and maximize FRET signal changes during rotation.

2. High-Speed Atomic Force Microscopy (HS-AFM):

• Methodology: Image surface-attached ATP synthase complexes at video rate (>10 frames/second).

• Analysis: Track topographical changes associated with c-ring rotation.

• Advantages: Allows direct visualization of rotational movements without requiring protein labeling.

• Implementation: Requires careful immobilization of the complex to expose the c-ring while maintaining its rotational freedom.

3. Gold Nanorod Probes with Optical Microscopy:

• Methodology: Attach gold nanorods to the c-ring and track their orientation using polarized light.

• Analysis: Monitor changes in light scattering as the nanorod rotates with the c-ring.

• Advantages: Provides high angular and temporal resolution with minimal photodamage.

• Implementation: Requires specific attachment of nanorods to the c-ring without impeding rotation.

4. Cryo-EM Classification Approaches:

• Methodology: Collect large datasets of ATP synthase particles and classify them based on c-ring rotational state.

• Analysis: Identify distinct conformational states representing different rotational positions, as demonstrated for Bacillus PS3 ATP synthase where three rotational states with integer step sizes of approximately 3, 4, and 3 c-subunits were observed .

• Advantages: Provides structural information at near-atomic resolution for each rotational state.

• Implementation: Requires sophisticated image processing and classification algorithms to resolve different rotational states.

5. Molecular Dynamics Simulations:

• Methodology: Build atomic models of the Shewanella ATP synthase based on homology to known structures (e.g., Bacillus PS3) and simulate proton movement and associated c-ring rotation.

• Analysis: Calculate energy landscapes, transition probabilities, and rotational trajectories.

• Advantages: Provides detailed atomic-level insights into the mechanism of rotation.

• Implementation: Requires significant computational resources and careful parameterization of the simulation system.

The combination of these techniques can provide complementary information about c-ring rotation, from structural snapshots of different rotational states to real-time dynamics of the rotational process. The choice of technique depends on the specific research question, available resources, and required temporal and spatial resolution.

How does the proton translocation mechanism in Shewanella halifaxensis ATP synthase compare to other bacterial species?

The proton translocation mechanism in ATP synthases involves a sophisticated pathway through the membrane domain that can vary between species. A comparative analysis reveals both conserved features and species-specific adaptations:

Conserved Elements of Proton Translocation:

All bacterial ATP synthases, including Shewanella halifaxensis, likely share fundamental features of the proton translocation mechanism:

• Two Half-Channel Architecture: The proton path involves two aqueous half-channels at the interface between subunit a and the c-ring, as described in the Bacillus PS3 ATP synthase structure . The cytoplasmic half-channel allows protons to exit to the cytoplasm, while the periplasmic half-channel provides access for protons from the periplasm to the c-ring.

• Conserved Residues: Key residues are highly conserved across bacterial species:

  • A critical glutamate residue on each c-subunit (e.g., Glu 56 in some bacteria) that undergoes protonation/deprotonation cycles .

  • A positively charged arginine in subunit a (e.g., Arg 169 in Bacillus PS3) that interacts with the deprotonated glutamate, facilitating proton release to the cytoplasmic half-channel .

• Rotational Mechanism: The sequential protonation of c-subunits from the periplasmic side and deprotonation to the cytoplasmic side drives the counter-clockwise rotation (viewed from F₁ toward F₀) of the c-ring .

Species-Specific Variations:

*Values predicted based on homology and bacterial ATP synthase conservation patterns, as specific data for S. halifaxensis is not directly provided in the search results.

Methodological Approaches for Comparative Studies:

• Structural Analysis:

  • Cryo-EM structures of Shewanella ATP synthase compared with known structures (like Bacillus PS3) .

  • Homology modeling based on conserved features.

• Mutational Studies:

  • Swap critical residues between species to determine their role in species-specific functions.

  • Create chimeric proteins with components from different bacterial ATP synthases.

• Functional Assays:

  • Compare proton pumping efficiency using pH measurements similar to those used for S. oneidensis .

  • Assess ATP synthesis rates under various conditions.

• Computational Approaches:

  • Molecular dynamics simulations to compare proton pathways.

  • Quantum mechanics/molecular mechanics calculations to understand energetics of proton transfer.

These comparative studies would help elucidate how Shewanella halifaxensis ATP synthase has evolved to function optimally in its specific environmental niche, potentially revealing adaptations related to its role in extracellular electron transfer processes that are characteristic of Shewanella species.

How does the ATP synthase activity in Shewanella halifaxensis relate to its unique extracellular electron transfer capabilities?

Shewanella species are known for their remarkable ability to perform extracellular electron transfer (EET), and their ATP synthase activity is intricately connected to this process through energy metabolism pathways:

Integration of ATP Synthesis and Extracellular Electron Transfer:

• Proton Motive Force Generation During EET:
  During extracellular electron transfer, Shewanella species transfer electrons to external acceptors through specialized membrane proteins. This process can generate a proton motive force (PMF) that drives ATP synthesis through ATP synthase . Studies with S. oneidensis have shown that PMF generation is critical for maintaining energy levels during EET processes.

• ATP Requirements for EET Processes:
  The extracellular electron transfer machinery requires ATP for assembly, maintenance, and in some cases, direct energization. The ATP synthase provides this energy currency, creating a reciprocal relationship between EET and ATP synthesis.

• Metabolic Adaptations:
  Shewanella species have evolved specialized metabolic pathways that couple lactate oxidation to both ATP production and electron transfer to external acceptors. This metabolic flexibility allows them to thrive in environments with variable electron acceptor availability .

Experimental Evidence and Research Approaches:

Studies with Shewanella oneidensis, a close relative of S. halifaxensis, have provided insights into the relationship between ATP synthase activity and EET:

• Enhanced EET with Supplemental PMF:
  Experiments with S. oneidensis expressing light-driven proton pumps demonstrated that additional PMF generation significantly enhanced EET efficiency. This finding indicates that PMF availability, which drives ATP synthesis, is often a limiting factor in EET processes .

• Transcriptomic Evidence:
  Transcriptome analysis revealed that genes encoding ATP synthase subunits (atpA-I) were significantly upregulated when additional PMF was available, suggesting tight coordination between PMF utilization for ATP synthesis and EET processes .

• Metabolic Measurements:
  Measurements of lactate consumption, acetate production, and NAD(H) levels showed that enhanced PMF led to increased metabolic flux, providing more electrons for the EET pathway .

Methodological Approaches for Studying This Relationship:

• Genetic Manipulation:

  • Create atpE (subunit c) mutants with altered properties and assess their impact on both ATP synthesis and EET rates.

  • Develop controlled expression systems to modulate ATP synthase levels and observe effects on EET.

• Bioenergetic Measurements:

  • Simultaneously monitor PMF (using fluorescent probes), ATP levels, and electron transfer rates to external acceptors.

  • Apply specific inhibitors of ATP synthase to decouple ATP synthesis from EET.

• Advanced Imaging:

  • Use correlative light and electron microscopy to visualize the spatial organization of ATP synthase relative to EET components in the Shewanella membrane.

  • Apply super-resolution microscopy to track dynamic interactions between energy-generating and electron-transferring complexes.

• Systems Biology Approaches:

  • Develop computational models that integrate ATP synthesis, PMF generation, and EET processes.

  • Apply metabolic flux analysis to quantify the distribution of energy and reducing equivalents between ATP synthesis and EET.

These research approaches would help elucidate how Shewanella halifaxensis has evolved its ATP synthase to support its exceptional capability for extracellular electron transfer, potentially revealing unique adaptations in the structure or regulation of ATP synthase subunit c that optimize its function in this specialized bacterial system.

What are the challenges in crystallizing or obtaining high-resolution structures of recombinant Shewanella halifaxensis ATP synthase subunit c?

Obtaining high-resolution structures of membrane proteins like ATP synthase subunit c presents significant challenges that require specialized approaches:

Key Technical Challenges:

• Protein Instability Outside the Membrane Environment:

  • The hydrophobic nature of subunit c makes it unstable when removed from the lipid bilayer.

  • The c-ring structure may dissociate during purification, compromising structural integrity.

• Crystallization Difficulties:

  • Detergent micelles surrounding the hydrophobic regions create heterogeneous particles that hinder crystal formation.

  • The limited polar surface area available for crystal contacts reduces crystallization probability.

• Conformational Heterogeneity:

  • ATP synthase exhibits multiple rotational states, as observed in Bacillus PS3 ATP synthase , creating a mixture of conformations that complicate structure determination.

  • The inherent flexibility required for function can prevent formation of well-ordered crystals.

• Expression and Purification Limitations:

  • Overexpression may lead to inclusion body formation or toxicity to host cells.

  • Purification may result in loss of essential lipids or cofactors needed for native structure.

Methodological Solutions:

• Cryo-EM Approach:

  • Single-particle cryo-EM has emerged as the method of choice for membrane protein structure determination, avoiding the need for crystallization.

  • Classification algorithms can sort particles into discrete rotational states, as demonstrated for Bacillus PS3 ATP synthase .

  • Sample preparation protocols using gentle detergents like glycol-diosgenin (GDN) at 0.02% concentration preserve complex integrity .

• Lipid Nanodisc Technology:

  • Reconstitute the c-subunit or c-ring into nanodiscs to provide a more native-like lipid environment.

  • This approach maintains protein stability and reduces conformational heterogeneity.

• Stabilizing Strategies:

  • Introduce disulfide bonds to lock the c-ring in a specific conformation.

  • Co-purify with binding partners (e.g., subunit a) to stabilize the complex.

  • Include stabilizing agents such as 6-aminocaproic acid and benzamidine throughout purification .

• Alternative Crystallization Approaches:

  • Lipidic cubic phase (LCP) crystallization, which provides a membrane-like environment.

  • Antibody fragment (Fab) co-crystallization to increase polar surface area.

  • Fusion protein strategies to enhance solubility and provide crystal contacts.

• Hybrid Structural Biology Approaches:

  • Combine lower-resolution cryo-EM of the intact complex with high-resolution structures of individual components.

  • Use molecular dynamics simulations to refine structures and understand dynamic aspects.

  • Apply solid-state NMR to obtain structural constraints in a lipid environment.

The successful structural determination of Bacillus PS3 ATP synthase using cryo-EM provides a valuable methodological template that could be adapted for Shewanella halifaxensis ATP synthase, focusing on careful protein preparation, stabilization, and the application of advanced image processing techniques to resolve different conformational states.

How can isotope labeling be used to study the function and dynamics of Shewanella halifaxensis ATP synthase subunit c?

Isotope labeling provides powerful approaches for investigating the structure, function, and dynamics of ATP synthase subunit c that complement conventional structural biology methods:

Strategic Isotope Labeling Approaches:

• Site-Specific Isotope Labeling for NMR Studies:

  • Incorporate ¹⁵N, ¹³C, or ²H at specific residues of interest, particularly those involved in proton translocation, such as the conserved glutamate residue.

  • Methodology: Use amber suppression technology with orthogonal aminoacyl-tRNA synthetase/tRNA pairs to incorporate isotope-labeled amino acids at specific positions.

  • Analysis: Perform solid-state NMR experiments to determine chemical shifts, which provide information about local electronic environment, hydrogen bonding, and protonation states.

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

  • Expose purified ATP synthase to D₂O buffer and monitor the rate of hydrogen/deuterium exchange over time.

  • Methodology: Quench the exchange reaction at various time points, digest the protein, and analyze peptides by mass spectrometry.

  • Analysis: Regions with rapid exchange are exposed or flexible, while regions with slow exchange are buried or involved in stable hydrogen bonds, providing insights into dynamics and solvent accessibility of different regions of subunit c.

• Heavy Oxygen (¹⁸O) Labeling for Proton Translocation Studies:

  • Label the water molecules with ¹⁸O and track oxygen exchange between water and the carboxyl group of the key glutamate residue.

  • Methodology: Use mass spectrometry to detect ¹⁸O incorporation into specific residues.

  • Analysis: Rate of oxygen exchange provides information about the protonation/deprotonation cycle of the key glutamate residue during ATP synthesis.

• Neutron Scattering with Selective Deuteration:

  • Deuterate specific regions of subunit c to enhance contrast in neutron scattering experiments.

  • Methodology: Express the protein in media containing D₂O and deuterated carbon sources, with strategic protonation of specific regions.

  • Analysis: Perform neutron diffraction or small-angle neutron scattering to obtain structural information complementary to X-ray or cryo-EM data, with particular sensitivity to hydrogen atom positions.

Experimental Protocol Design Table:

Data Analysis and Interpretation:

• Correlation with Functional States:

  • Combine isotope labeling studies with functional assays (ATP synthesis, proton pumping) to correlate structural dynamics with function.

  • Compare results obtained under different conditions (pH, membrane potential) to understand how these factors affect the conformation and dynamics of subunit c.

• Integration with Computational Models:

  • Use experimental constraints from isotope labeling studies to refine and validate molecular dynamics simulations.

  • Develop models that explain how protonation changes drive conformational changes and ultimately c-ring rotation.

• Comparative Analysis:

  • Apply the same isotope labeling approaches to wild-type and mutant proteins to understand how specific residues contribute to function.

  • Compare results from Shewanella halifaxensis ATP synthase with those from other bacterial species to identify species-specific features.

These isotope labeling approaches provide unique insights into the molecular mechanism of ATP synthase function that are difficult to obtain through other methods, particularly regarding proton dynamics, conformational changes, and the coupling between proton translocation and c-ring rotation.

What are the emerging technologies that could advance our understanding of Shewanella halifaxensis ATP synthase function in extracellular electron transfer?

The intersection of ATP synthase function and extracellular electron transfer (EET) in Shewanella represents a frontier for bioenergetics research. Several emerging technologies could significantly advance our understanding of this relationship:

1. Advanced Imaging Technologies:

• Cryo-Electron Tomography (Cryo-ET) with Focused Ion Beam Milling:
  This technique allows visualization of ATP synthase in its native cellular context, revealing its spatial relationship with EET components. Recent advances in sub-tomogram averaging can achieve near-atomic resolution of membrane protein complexes in situ.

• Super-Resolution Microscopy with Single-Molecule Tracking:
  Techniques like PALM, STORM, or MINFLUX can track individual ATP synthase molecules in living Shewanella cells, revealing their dynamics and potential co-localization with electron transfer components with nanometer precision.

• Correlative Light and Electron Microscopy (CLEM):
  This approach combines fluorescence microscopy of labeled ATP synthase with electron microscopy of the same sample, providing both functional and structural information in the cellular context.

2. Advanced Spectroscopic Methods:

• Time-Resolved FTIR Spectroscopy:
  This can detect conformational changes and protonation states of key residues in ATP synthase during catalysis with microsecond time resolution, particularly valuable for studying the proton-binding glutamate residue.

• Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling:
  By introducing spin labels at strategic positions in subunit c, researchers can measure distances and detect conformational changes during ATP synthesis coupled to EET.

• Surface-Enhanced Raman Spectroscopy (SERS):
  This technique can provide chemical information about ATP synthase and electron transfer components at the membrane-electrode interface with single-molecule sensitivity.

3. Synthetic Biology and Bioengineering Approaches:

• Optogenetic Control of ATP Synthase Activity:
  Designing light-sensitive domains into ATP synthase components would allow precise temporal control of ATP production, enabling researchers to study how rapid changes in cellular energy state affect EET.

• Genetically Encoded Biosensors:
  Developing fluorescent sensors for ATP, PMF, and electron flow would allow real-time monitoring of these parameters in living cells, revealing the dynamic interplay between ATP synthesis and EET.

• Cell-Free Reconstituted Systems:
  Creating minimal synthetic systems that couple ATP synthase activity with electron transfer components in artificial membranes or nanoreactors would allow precise manipulation of individual components.

4. Computational and Systems Biology Methods:

• Multi-scale Molecular Simulation:
  Combining quantum mechanical calculations of proton transfer with molecular dynamics of protein conformational changes and coarse-grained models of membrane processes can provide a comprehensive understanding of how ATP synthase couples to EET.

• Metabolic Flux Analysis with Stable Isotope Labeling:
  Using ¹³C-labeled substrates combined with metabolomics can quantify how carbon and energy flow through different pathways during EET, revealing the metabolic context of ATP synthase function.

• Machine Learning for Pattern Recognition in Complex Datasets:
  Applying machine learning to analyze correlations between ATP synthesis rates, electron transfer rates, and environmental conditions could reveal previously unrecognized patterns and regulatory mechanisms.

5. Novel Biophysical Techniques:

• Single-Molecule Force Spectroscopy:
  Using techniques like magnetic tweezers or acoustic force spectroscopy to apply controlled torque to the ATP synthase rotor can provide insights into the mechanical properties of the c-ring and its response to PMF.

• Nanopore Technology:
  Reconstituting ATP synthase into artificial nanopores allows precise electrical measurements of proton translocation with single-molecule resolution.

• Microfluidic Devices with Integrated Electrodes:
  These devices can simultaneously control the chemical environment, measure electrical current from EET, and monitor ATP production, providing integrated information on the coupling between these processes.

The integration of these emerging technologies promises to provide unprecedented insights into how Shewanella halifaxensis ATP synthase contributes to the organism's unique ability to transfer electrons to extracellular acceptors, potentially leading to new applications in bioelectrochemical systems and microbial fuel cells .

How might engineered variants of Shewanella halifaxensis ATP synthase subunit c be utilized in synthetic biology applications?

Engineered variants of Shewanella halifaxensis ATP synthase subunit c offer promising opportunities for synthetic biology applications, particularly in bioenergetics, bioelectrochemical systems, and biotechnology:

1. Enhanced Bioelectrochemical Systems:

Engineered ATP synthase c-subunits could significantly improve microbial fuel cells and bioelectrochemical systems by:

• Optimized Proton Translocation Efficiency:
  Modifying the conserved glutamate residue or surrounding amino acids to alter its pKa could enhance proton binding/release kinetics, potentially increasing the efficiency of energy conversion from the proton motive force to ATP.

• Altered Stoichiometry c-rings:
  Engineering c-rings with different numbers of subunits would change the H⁺/ATP ratio, allowing fine-tuning of the balance between ATP production and electron transfer rates. This approach could be particularly valuable in optimizing Shewanella-based microbial fuel cells.

• Light-Responsive ATP Synthase Systems:
  Creating hybrid systems combining engineered c-subunits with light-driven proton pumps (similar to the strain cR-1 system described for S. oneidensis ) could enable light-controlled ATP production and enhanced extracellular electron transfer in bioelectrochemical applications.

2. Novel Biosensors and Diagnostic Tools:

• PMF-Responsive Biosensors:
  Engineered c-subunits with incorporated fluorescent reporters could serve as sensitive detectors of changes in proton motive force, useful for screening antimicrobial compounds that affect bacterial bioenergetics.

• ATP Production Monitoring Systems:
  Coupling modified ATP synthase complexes with luminescent reporters would allow real-time monitoring of ATP synthesis rates in response to various stimuli, useful for high-throughput screening applications.

• Electrochemical Biosensors:
  ATP synthase variants incorporated into electrode surfaces could detect compounds that affect proton translocation, providing a new class of highly sensitive biosensors for environmental monitoring.

3. Biomimetic Energy Conversion Devices:

• Nanoscale Energy Converters:
  Immobilized engineered c-rings on synthetic membranes or electrodes could convert electrical potential into mechanical work or chemical synthesis, mimicking the natural function of ATP synthase but in artificial systems.

• Hybrid Biological-Electronic Interfaces:
  Modified ATP synthase c-subunits could serve as molecular bridges between biological systems and electronic components, enabling direct conversion between biological energy currencies (ATP, PMF) and electrical energy.

• Artificial Photosynthetic Systems:
  Combining engineered ATP synthase with synthetic light-harvesting components could create biomimetic systems for solar energy conversion and storage.

4. Bioproduction of High-Energy Compounds:

• Enhanced Metabolic Engineering Platforms:
  Engineered ATP synthase variants could be introduced into production strains to optimize energy metabolism, potentially increasing yields of target compounds by balancing ATP production with other metabolic needs.

• ATP-Driven Synthesis of Valuable Compounds:
  Modified ATP synthases could be engineered to couple ATP hydrolysis to the synthesis of high-value chemicals, essentially running the enzyme in reverse with altered specificity.

• Controlled Fermentation Systems:
  Engineered ATP synthase variants with altered regulation could help maintain optimal intracellular ATP levels during fermentation processes, potentially improving product yields and process stability.

Implementation Strategies and Considerations:

The successful implementation of these applications would build upon the fundamental understanding of proton translocation in bacterial ATP synthases and the demonstrated feasibility of enhancing extracellular electron transfer through manipulation of proton motive force generation , ultimately leading to new technologies at the interface of synthetic biology and bioelectrochemistry.