Recombinant Pan troglodytes Cytochrome c oxidase subunit 2 (MT-CO2)

What is MT-CO2 and what is its role in cellular respiration?

MT-CO2 (Cytochrome c oxidase subunit 2) is a critical component of the respiratory chain in mitochondria. It forms part of Complex IV (cytochrome c oxidase), which catalyzes the reduction of oxygen to water - the final step in the electron transport chain. Specifically, MT-CO2 functions by transferring electrons from cytochrome c via its binuclear copper A center to the bimetallic center of the catalytic subunit 1 . This electron transfer activity is essential for establishing the proton gradient that drives ATP synthesis. In Pan troglodytes (chimpanzees), MT-CO2 is encoded by the mitochondrial genome and shares high sequence homology with human MT-CO2, making it valuable for evolutionary and functional comparative studies.

What is the protein structure of Pan troglodytes MT-CO2?

Pan troglodytes MT-CO2 is a multi-pass membrane protein located in the inner mitochondrial membrane. The protein consists of 228 amino acids with a molecular weight of approximately 26 kDa . The protein contains several transmembrane domains that anchor it within the mitochondrial inner membrane, with specific regions extending into both the intermembrane space and the mitochondrial matrix. Key functional domains include the copper-binding sites that form the copper A center, which is essential for electron transfer activity. The three-dimensional structure reveals a complex folding pattern that positions the copper centers optimally for interaction with both cytochrome c and the catalytic core of the enzyme.

How does the amino acid sequence of Pan troglodytes MT-CO2 compare to human MT-CO2?

The amino acid sequence of Pan troglodytes MT-CO2 shows remarkable conservation when compared to human MT-CO2, reflecting their close evolutionary relationship. The two proteins share approximately 98-99% sequence identity, with only a few amino acid substitutions. These substitutions are primarily located in non-critical regions of the protein, preserving the functional domains essential for electron transfer and catalytic activity. The high degree of conservation highlights the evolutionary constraints on this protein due to its essential role in cellular respiration. The specific sequence for Pan troglodytes MT-CO2 begins with "MATWANLGLQDSSSPLMEQL..." and continues through the 228 amino acid positions .

What expression systems are optimal for producing recombinant Pan troglodytes MT-CO2?

Several expression systems can be used for producing recombinant Pan troglodytes MT-CO2, each with distinct advantages depending on research objectives:

For functional studies requiring proper folding and copper incorporation, insect cell or mammalian expression systems are recommended despite lower yields. For structural studies requiring larger quantities, E. coli systems with subsequent refolding protocols may be more appropriate .

What purification strategies yield the highest purity for recombinant Pan troglodytes MT-CO2?

Purification of recombinant Pan troglodytes MT-CO2 typically involves a multi-step approach to achieve high purity (≥85%) :

• Initial Extraction: For membrane proteins like MT-CO2, detergent-based extraction is critical. Mild detergents such as n-dodecyl β-D-maltoside (DDM) or digitonin are preferred to maintain protein integrity.

• Affinity Chromatography: Utilizing N-terminal or C-terminal tags (His-tag, FLAG-tag, etc.) enables efficient capture of the target protein. Immobilized metal affinity chromatography (IMAC) with nickel or cobalt resins is particularly effective for His-tagged constructs.

• Ion Exchange Chromatography: This intermediate step removes contaminants based on charge differences. MT-CO2 typically exhibits a slightly acidic isoelectric point, making anion exchange chromatography (Q-Sepharose) suitable at neutral pH.

• Size Exclusion Chromatography: As a final polishing step, gel filtration separates aggregates and provides the protein in a well-defined oligomeric state.

• Quality Control: SDS-PAGE analysis confirms purity of ≥85%, while spectroscopic methods verify copper incorporation by examining the characteristic absorption peaks.

For researchers requiring exceptional purity for crystallography or other high-resolution structural studies, additional chromatographic steps or selective precipitation methods may be necessary.

How can functional activity of recombinant Pan troglodytes MT-CO2 be verified after purification?

Verifying the functional activity of recombinant Pan troglodytes MT-CO2 after purification is essential to ensure that the protein maintains its native properties. Multiple complementary approaches are recommended:

• Spectroscopic Analysis: UV-visible spectroscopy can confirm proper copper incorporation by measuring absorption peaks characteristic of the copper A center (typically around 480-500 nm and 830 nm).

• Electron Transfer Assay: The primary function of MT-CO2 is electron transfer from cytochrome c. This can be assessed using reduced cytochrome c as an electron donor and monitoring the oxidation rate spectrophotometrically at 550 nm.

• Oxygen Consumption Measurements: When incorporated into proteoliposomes with other subunits of cytochrome c oxidase, oxygen consumption rates can be measured using oxygen electrodes or fluorescence-based oxygen sensors.

• Copper Content Analysis: Atomic absorption spectroscopy or inductively coupled plasma mass spectrometry (ICP-MS) can quantify copper content, which should approach 2 copper atoms per MT-CO2 molecule in properly folded protein.

• Circular Dichroism (CD) Spectroscopy: This technique assesses secondary structure integrity, providing evidence that the recombinant protein has folded correctly.

A combination of these approaches provides comprehensive validation of functional integrity before proceeding with further experimental applications.

How does Pan troglodytes MT-CO2 inform our understanding of primate evolution?

Pan troglodytes MT-CO2 serves as a valuable molecular marker for understanding primate evolution due to several key characteristics:

• Mitochondrial Inheritance: As a mitochondrial gene, MT-CO2 is maternally inherited without recombination, providing a clear evolutionary lineage.

• Evolutionary Rate: MT-CO2 evolves at an intermediate rate compared to other mitochondrial genes, making it useful for resolving relationships among closely related primate species.

• Functional Constraints: Comparative analysis reveals regions under strong purifying selection (functional domains) versus regions experiencing relaxed selection or positive selection.

Analysis of sequence variations between Pan troglodytes MT-CO2 and other primate species has contributed to molecular clock estimates suggesting that the human-chimpanzee divergence occurred approximately 5-7 million years ago. Furthermore, specific amino acid substitutions in MT-CO2 across primate lineages correlate with metabolic adaptations, potentially reflecting environmental adaptations throughout evolutionary history.

The conserved nature of copper-binding sites across all primate MT-CO2 sequences underscores the functional importance of these regions, while variations in transmembrane domains may relate to membrane environment adaptations or interactions with nuclear-encoded subunits that co-evolved with MT-CO2.

What approaches are most effective for analyzing selective pressures on Pan troglodytes MT-CO2?

Analyzing selective pressures on Pan troglodytes MT-CO2 requires sophisticated computational and experimental approaches:

Effective analysis typically combines multiple approaches. For example, computational prediction of sites under selection can be followed by experimental validation through site-directed mutagenesis and functional assays. When analyzing MT-CO2, particular attention should be paid to:

• Interaction sites with nuclear-encoded subunits (potential co-evolution)

• Regions involved in proton translocation

• Copper-binding domains

• Transmembrane regions that may adapt to different lipid environments

These analyses have revealed that while MT-CO2 is generally under strong purifying selection due to its essential function, specific sites show evidence of positive selection that may relate to metabolic adaptations in different primate lineages.

How can recombinant Pan troglodytes MT-CO2 be used to study mitochondrial disorders?

Recombinant Pan troglodytes MT-CO2 provides a valuable tool for studying mitochondrial disorders through several experimental approaches:

• Comparative Functional Studies: Human MT-CO2 mutations associated with mitochondrial disorders can be introduced into the chimpanzee ortholog to assess functional conservation and species-specific effects. This approach helps distinguish pathogenic mutations from benign polymorphisms.

• Biochemical Characterization: Purified recombinant MT-CO2 enables detailed biochemical studies of enzymatic properties, including:

  • Electron transfer kinetics

  • Oxygen reduction rates

  • Proton pumping efficiency

  • Sensitivity to inhibitors

• Structural Impact Analysis: By introducing disease-associated mutations into recombinant Pan troglodytes MT-CO2, researchers can study structural perturbations using techniques such as circular dichroism, thermal stability assays, and limited proteolysis.

• Protein-Protein Interaction Studies: Co-immunoprecipitation and surface plasmon resonance using recombinant MT-CO2 can identify altered interactions with other respiratory complex components, potentially revealing mechanisms of disease pathogenesis.

• Cellular Models: Expressing wild-type or mutant recombinant MT-CO2 in cell lines with depleted endogenous MT-CO2 allows assessment of functional complementation and cellular phenotypes like ATP production, reactive oxygen species generation, and apoptotic susceptibility.

This comparative approach between human and chimpanzee MT-CO2 is particularly valuable because it leverages the high sequence similarity to identify critical functional determinants while providing enough evolutionary distance to highlight adaptively important regions.

What methodologies are recommended for studying protein-protein interactions involving Pan troglodytes MT-CO2?

Studying protein-protein interactions involving Pan troglodytes MT-CO2 requires specialized approaches due to its membrane-embedded nature and complex integration within the respiratory chain:

• Crosslinking Mass Spectrometry (XL-MS): This technique uses chemical crosslinkers to capture transient interactions, followed by mass spectrometry to identify interaction partners and contact points. Specialized crosslinkers like DSS (disuccinimidyl suberate) for lysine residues or EDC for carboxyl-amine interactions are particularly effective.

• Co-Immunoprecipitation with Intact Mitochondrial Complexes: Using antibodies against tagged recombinant MT-CO2 can pull down intact interaction partners. Mild detergents like digitonin preserve supercomplexes for analysis of higher-order interactions.

• Blue Native PAGE: This non-denaturing electrophoresis technique preserves protein complexes and can be followed by a second-dimension SDS-PAGE to resolve individual components, revealing the integration of MT-CO2 within larger assemblies.

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics, purified recombinant MT-CO2 can be immobilized on sensor chips with controlled orientation using terminal tags, allowing measurement of binding affinities with other purified components.

• Proximity Labeling Methods: Techniques like BioID or APEX2, where MT-CO2 is fused to a proximity-dependent labeling enzyme, can identify the protein neighborhood in living cells, capturing even weak or transient interactions.

• Computational Prediction and Molecular Docking: In silico approaches can predict interaction interfaces based on structural models, which can then guide experimental validation through site-directed mutagenesis.

When reporting interaction data, it's essential to distinguish direct physical interactions from functional associations and to validate key findings using multiple complementary methods.

What are the critical factors for successful reconstitution of recombinant Pan troglodytes MT-CO2 into proteoliposomes?

Successful reconstitution of recombinant Pan troglodytes MT-CO2 into proteoliposomes requires careful consideration of multiple factors to maintain functional integrity:

• Lipid Composition: The lipid environment dramatically affects MT-CO2 function. A mixture mimicking the mitochondrial inner membrane is optimal, typically including:

  • 20-30% cardiolipin

  • 40-50% phosphatidylcholine

  • 20-30% phosphatidylethanolamine

  • Trace amounts of phosphatidylinositol

• Protein-to-Lipid Ratio: The optimal ratio typically ranges from 1:50 to 1:200 (w/w) depending on the experimental goals. Higher protein densities may be used for functional studies, while lower densities minimize protein-protein interactions for single-molecule studies.

• Detergent Removal Method: The technique used to remove detergent significantly impacts reconstitution efficiency:

  • Dialysis: Gentle but time-consuming (24-48 hours)

  • Bio-Beads: Faster (2-4 hours) but may adsorb some lipids

  • Dilution: Simple but results in low protein incorporation efficiency

• Buffer Conditions:

  • pH: Typically 7.2-7.4 to mimic physiological conditions

  • Ionic strength: 100-150 mM KCl or NaCl

  • Divalent cations: 1-5 mM Mg²⁺ to stabilize membrane structure

• Temperature: Reconstitution at temperatures slightly above the lipid phase transition temperature improves protein incorporation.

• Orientation Control: Techniques such as pH gradients during reconstitution can help achieve a more uniform protein orientation, which is critical for functional studies of proton pumping.

• Quality Control Assessments:

  • Freeze-fracture electron microscopy to verify protein incorporation

  • Dynamic light scattering to confirm vesicle size distribution

  • Fluorescence-based assays to verify membrane integrity

  • Protease protection assays to determine protein orientation

Successful reconstitution typically yields proteoliposomes with 70-80% of MT-CO2 in the correct orientation, allowing for accurate assessment of electron transfer activities and proton pumping efficiency.

How can cryo-electron microscopy be optimized for structural studies of recombinant Pan troglodytes MT-CO2?

Cryo-electron microscopy (cryo-EM) offers unprecedented opportunities for structural studies of membrane proteins like Pan troglodytes MT-CO2, but requires careful optimization:

• Sample Preparation Considerations:

  • Protein Purity: ≥95% homogeneity is essential, with additional size-exclusion chromatography immediately before grid preparation

  • Concentration: Typically 2-5 mg/mL for isolated MT-CO2, or 1-2 mg/mL for complete cytochrome c oxidase complex

  • Detergent Selection: LMNG (lauryl maltose neopentyl glycol) or GDN (glyco-diosgenin) often yield better results than traditional detergents for maintaining protein stability while minimizing background in images

• Grid Optimization:

  • Support Films: Ultrathin carbon or graphene oxide supports improve particle distribution

  • Hole Size and Spacing: Quantifoil R1.2/1.3 or R2/2 grids typically work well

  • Glow Discharge Parameters: 30-45 seconds at 15-20 mA for optimal hydrophilicity

• Vitrification Conditions:

  • Blotting Time: Usually 3-5 seconds, but requires empirical optimization

  • Blotting Force: Typically -5 to -2 on Vitrobot systems

  • Temperature/Humidity: 4°C and 100% humidity minimize sample evaporation

• Data Collection Strategy:

  • Voltage: 300 kV provides optimal contrast for membrane proteins

  • Defocus Range: -0.8 to -2.5 μm, collected in 0.3 μm increments

  • Total Electron Dose: 50-60 e⁻/Ų distributed across 40-50 frames to minimize radiation damage

  • Magnification: Yielding 0.8-1.2 Å/pixel for detailed structural analysis

• Processing Considerations:

  • Particle Picking: Reference-free picking followed by 2D classification to identify well-resolved particles

  • Classification Strategy: Focused classification around the MT-CO2 region may be necessary if studying the entire complex

  • Mask Design: Soft masks that include the detergent micelle but focus refinement on the protein

• Validation Approaches:

  • Resolution Assessment: Gold-standard FSC with 0.143 criterion

  • Model Validation: EMRinger scores, MolProbity statistics, and Q-scores for local resolution estimation

  • Functional Correlation: Structural features should be validated against biochemical/mutational data

Following these guidelines, researchers have achieved resolutions of 2.5-3.5 Å for cytochrome c oxidase complexes, revealing detailed insights into the structure-function relationships of MT-CO2 and its interactions with other subunits.

What strategies are recommended for analyzing the effects of post-translational modifications on Pan troglodytes MT-CO2?

Analyzing post-translational modifications (PTMs) of Pan troglodytes MT-CO2 requires an integrated approach combining targeted enrichment, high-resolution detection methods, and functional validation:

• Identification of PTMs:

• Functional Characterization Approaches:

  • Site-directed mutagenesis: Substituting modified residues with non-modifiable analogs (e.g., Ser→Ala for phosphorylation sites)

  • Enzyme activity assays: Measuring electron transfer rates before and after treatment with modifying/demodifying enzymes

  • Protein-protein interaction studies: Assessing how PTMs affect assembly into the cytochrome c oxidase complex

  • Thermal shift assays: Determining impacts on protein stability

  • Molecular dynamics simulations: Predicting structural consequences of modifications

• Physiological Context Investigation:

  • Stress conditions: Oxidative stress, hypoxia, and metabolic perturbations can induce specific PTMs

  • Developmental stages: Comparing PTM profiles across different developmental stages

  • Tissue specificity: Examining variations in PTM patterns across different tissues

  • Comparative analysis: Contrasting PTM patterns between human and chimpanzee MT-CO2 to identify conserved regulatory mechanisms

• Quantitative Analysis:

  • SILAC or TMT labeling for quantitative mass spectrometry

  • Parallel reaction monitoring (PRM) for targeted quantification of specific modified peptides

  • Western blotting with modification-specific antibodies for relative quantification

These approaches have revealed that Pan troglodytes MT-CO2 undergoes several PTMs including phosphorylation (particularly at serine residues), acetylation, oxidative modifications of cysteine and methionine residues, and in some cases ubiquitination. These modifications appear to regulate enzyme activity, assembly, and degradation under different physiological conditions.

How can Pan troglodytes MT-CO2 be used in ancestral sequence reconstruction studies?

Pan troglodytes MT-CO2 serves as a valuable reference point in ancestral sequence reconstruction (ASR) studies, offering insights into the evolution of mitochondrial function in primates:

• Methodological Approach for ASR Using MT-CO2:

  a. Sequence Collection:

  • Compile MT-CO2 sequences from diverse primate taxa (minimum 15-20 species across major lineages)

  • Include outgroups (non-primate mammals) to root the phylogeny

  • Ensure sequence quality and accurate annotation

  b. Phylogenetic Analysis:

  • Construct robust phylogenetic trees using maximum likelihood or Bayesian methods

  • Test multiple evolutionary models (JTT, WAG, LG for proteins; GTR, HKY for nucleotides)

  • Validate tree topology against established primate phylogeny

  c. Ancestral State Reconstruction:

  • Implement empirical Bayes or maximum likelihood methods

  • Calculate posterior probabilities for each ancestral amino acid

  • Consider alternative reconstructions for positions with ambiguity (PP < 0.8)

  d. Resurrection and Functional Testing:

  • Synthesize reconstructed ancestral MT-CO2 sequences

  • Express in suitable systems (bacterial, yeast, or mammalian cells)

  • Compare biochemical properties to extant proteins

• Key Research Applications:

  a. Detecting Adaptive Evolution:

  • Identify lineage-specific shifts in selection pressure

  • Correlate functional changes with environmental adaptations

  • Test hypotheses about metabolic evolution in primates

  b. Functional Divergence Analysis:

  • Measure changes in catalytic efficiency, substrate specificity, or stability

  • Identify critical amino acid substitutions driving functional shifts

  • Map changes to 3D structure to understand mechanistic basis

  c. Molecular Clock Calibration:

  • Use fossil-calibrated divergence times to estimate substitution rates

  • Test for rate heterogeneity across primate lineages

  • Refine molecular dating of key evolutionary events

• Technical Considerations:

  a. Uncertainty Handling:

  • Generate multiple plausible ancestral sequences for ambiguous nodes

  • Test all alternative reconstructions experimentally

  • Implement Bayesian sampling approaches to account for phylogenetic uncertainty

  b. Functional Expression Challenges:

  • Design chimeric constructs with extant sequences for membrane insertion

  • Co-express with appropriate nuclear-encoded subunits

  • Optimize codon usage for expression system

Research using this approach has revealed that several key substitutions in MT-CO2 occurred during early hominid evolution, potentially relating to changes in metabolic demands associated with increased brain size and altered locomotive patterns. These studies provide a molecular window into the metabolic adaptations that accompanied human evolution.

What approaches can resolve expression and solubility issues with recombinant Pan troglodytes MT-CO2?

Recombinant expression of membrane proteins like Pan troglodytes MT-CO2 frequently encounters solubility challenges. Here are systematic approaches to troubleshoot and optimize expression:

• Expression System Optimization:

• Extraction and Solubilization Strategies:

  a. Detergent Screening:

  • Mild non-ionic detergents (DDM, LMNG) preserve function but may have lower efficiency

  • Zwitterionic detergents (LDAO, Fos-Choline) offer higher extraction efficiency but may destabilize the protein

  • Systematic screening using a panel of 8-12 detergents at various concentrations (0.5-2% w/v)

  b. Solubilization Conditions:

  • Buffer composition: Test various pH values (6.5-8.0) and salt concentrations (100-500 mM)

  • Additives: Glycerol (5-10%), specific lipids (0.1-0.5 mg/mL), or stabilizing agents like cholesteryl hemisuccinate

  • Temperature: 4°C for most proteins, but room temperature may be more effective for some detergents

  c. Alternative Approaches:

  • Amphipol substitution after initial detergent extraction

  • Nanodisc incorporation for improved stability

  • Styrene maleic acid lipid particles (SMALPs) for detergent-free extraction

• Refolding Strategies (if inclusion bodies form):

  • Gradual dialysis to remove denaturants

  • On-column refolding during purification

  • Pulse dilution methods to prevent aggregation

• Validation of Proper Folding:

  • Spectroscopic methods to verify copper center formation

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to evaluate structural integrity

Implementing these strategies systematically, beginning with expression system optimization and followed by extraction condition screening, has successfully improved yields of functional MT-CO2 from <0.5 mg/L to 2-5 mg/L in optimized systems.

How can researchers address challenges in functional reconstitution of Pan troglodytes MT-CO2?

Functional reconstitution of Pan troglodytes MT-CO2, either alone or as part of the complete cytochrome c oxidase complex, presents several challenges that can be systematically addressed:

• Activity Loss During Purification:

  a. Metal Center Integrity:

  • Supplement buffers with 5-10 μM copper to prevent loss from copper centers

  • Avoid chelating agents like EDTA in purification buffers

  • Verify copper content using atomic absorption spectroscopy before reconstitution

  b. Oxidative Damage:

  • Include reducing agents (1-5 mM β-mercaptoethanol or 1 mM DTT) in all buffers

  • Work under nitrogen atmosphere for sensitive steps

  • Add antioxidants like 0.05-0.1 mM ascorbate to storage buffers

  c. Lipid Requirements:

  • Maintain specific lipids (particularly cardiolipin) during purification

  • Add 0.1-0.5 mg/mL lipid mixtures to extraction and purification buffers

  • Use lipid-detergent mixed micelles for intermediate stabilization

• Reconstitution Efficiency Challenges:

  a. Methodological Optimization:

  • Compare detergent removal methods (dialysis vs. Bio-Beads vs. cyclodextrin)

  • Optimize protein:lipid ratios (test range from 1:50 to 1:500 w/w)

  • Control rate of detergent removal (slow removal often yields better results)

  b. Orientation Control:

  • Apply pH gradients during reconstitution to bias orientation

  • Use asymmetric lipid compositions in the two leaflets

  • Validate orientation using accessibility assays with membrane-impermeable reagents

  c. Functional Assembly:

  • For complete complex reconstitution, assemble subunits in specific order

  • Pre-form subcomplexes before final reconstitution

  • Include assembly factors identified in native systems

• Activity Measurement Troubleshooting:

  a. Electron Transfer Activity:

  • If low activity, verify reduced cytochrome c quality (>95% reduction)

  • Optimize buffer conditions (150-200 mM KCl, pH 7.2-7.4)

  • Add catalase (100-200 U/mL) to remove hydrogen peroxide that may form

  b. Proton Pumping:

  • Use pH-sensitive fluorescent dyes (ACMA, pyranine) to monitor proton movement

  • Ensure vesicle integrity using carboxyfluorescein leakage assays

  • Create sufficient driving force with appropriate ionophores (valinomycin)

  c. Respiration Measurements:

  • Calibrate oxygen electrodes immediately before use

  • Include regenerating systems for electron donors

  • Control for auto-oxidation rates in all measurements

• Troubleshooting Workflow:

  • Begin with spectroscopic verification of copper centers

  • Progress to simple electron transfer assays

  • Advance to more complex coupled assays only after verifying basic function

  • Compare activities to published values for mammalian cytochrome c oxidase

Following this systematic approach helps identify the specific stage at which functional loss occurs and allows targeted interventions to preserve activity through the reconstitution process.

What are the current frontiers in Pan troglodytes MT-CO2 research?

Research on Pan troglodytes MT-CO2 continues to advance our understanding of mitochondrial function, evolution, and disease mechanisms. Current frontier areas include:

• Structural Biology Innovations: Cryo-electron microscopy has recently enabled visualization of the complete respiratory supercomplex ("respirasome") containing MT-CO2 at near-atomic resolution. These structures reveal previously unknown interaction interfaces and conformational changes during the catalytic cycle. Ongoing research aims to capture additional functional states and resolve dynamics using time-resolved cryo-EM.

• Evolutionary Medicine Applications: Comparative studies between human and chimpanzee MT-CO2 are illuminating the molecular basis of species-specific metabolic adaptations. These studies have identified critical residues that may explain differences in mitochondrial efficiency and susceptibility to oxidative damage, with implications for understanding human-specific diseases and aging processes.

• Mitochondrial Replacement Therapy: Research on MT-CO2 compatibility between species is informing ethical and technical discussions about mitochondrial replacement therapies. The high sequence similarity between human and chimpanzee MT-CO2 makes it a valuable model for understanding nuclear-mitochondrial compatibility issues.

• Systems Biology Integration: Network-based approaches are revealing how MT-CO2 functions within the broader cellular context, including its roles in apoptosis signaling, reactive oxygen species management, and cross-talk with nuclear gene expression. Multi-omics studies are mapping how MT-CO2 variants influence mitochondrial and cellular physiology.

• Synthetic Biology Applications: Engineered variants of MT-CO2 are being developed as biosensors for cellular oxygen levels and as components of artificial electron transport chains. These applications leverage the fundamental electron transfer capabilities of MT-CO2 for biotechnological purposes.