Recombinant Pseudalopex sechurae Cytochrome c oxidase subunit 2 (MT-CO2)

What is Cytochrome c oxidase subunit 2 (MT-CO2) and what is its function?

Cytochrome c oxidase subunit 2 (MT-CO2) is one of the core components of mitochondrial Cytochrome c oxidase (Cco), which functions as the terminal enzyme complex (Complex IV) in the electron transport chain. MT-CO2 contains a dual core CuA active site that plays a crucial role in the physiological process of cellular respiration. This protein is encoded by the mitochondrial DNA and serves as one of the three mtDNA-encoded subunits of respiratory complex IV . The primary function of MT-CO2 is to accept electrons from cytochrome c and transfer them through its copper center to other subunits of the complex, ultimately contributing to the reduction of oxygen to water. This process is essential for generating the proton gradient that drives ATP synthesis, making MT-CO2 vital for cellular energy production in aerobic organisms.

What is the structural composition of Pseudalopex sechurae MT-CO2?

Pseudalopex sechurae (Sechuran desert fox) MT-CO2 is a full-length protein comprising 227 amino acids . The protein has a molecular mass of approximately 25.6 kDa, similar to MT-CO2 proteins from other mammalian species . The structural elements include:

• N-terminal domain containing two transmembrane alpha-helices that anchor the protein in the mitochondrial inner membrane

• A hydrophilic domain containing the functional CuA center

• A binuclear copper center (CuA) located in a conserved cysteine loop, specifically at positions 196 and 200, with an additional conserved histidine at position 204

• The beginning of its amino acid sequence is MAYPFQLGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKLTHTSTMD

The protein structure includes both membrane-spanning regions and functional domains that project into the intermembrane space where electron transfer occurs.

How is recombinant Pseudalopex sechurae MT-CO2 protein typically produced?

Recombinant Pseudalopex sechurae MT-CO2 protein is typically produced using bacterial expression systems, with E. coli being the preferred host . The process involves:

• Gene synthesis or cloning of the MT-CO2 coding sequence (1-227 amino acids)

• Insertion into an expression vector with an N-terminal His-tag for purification purposes

• Transformation into a suitable E. coli strain for protein expression

• Induction of protein expression using IPTG in the bacterial culture

• Cell lysis to release the recombinant protein

• Purification using affinity chromatography with Ni²⁺-NTA agarose, leveraging the His-tag

• Final processing to obtain the purified protein, often as a lyophilized powder for stability

This expression system typically yields a recombinant protein of approximately 44 kDa (including fusion tags), with concentrations around 50 μg/mL after purification .

What factors affect the enzymatic activity of recombinant MT-CO2, and how can activity be measured?

The enzymatic activity of recombinant MT-CO2 is influenced by several factors that researchers must consider:

• Copper incorporation: The dual core CuA active site is essential for electron transfer activity. Proper formation of this metal center is critical for function .

• Protein folding: Correct tertiary structure is necessary for the positioning of copper-binding residues and formation of the active site.

• Environmental factors: Temperature, pH, ionic strength, and presence of detergents can all affect the catalytic efficiency of the protein.

• Substrate availability: The oxidation of cytochrome c by MT-CO2 depends on the redox state of the substrate.

• Modulators: Compounds like allyl isothiocyanate (AITC) can influence the catalytic activity of the enzyme .

Measurement methods include:

• Spectrophotometric assays: Monitoring the oxidation of reduced cytochrome c at 550 nm, which shows a decrease in absorbance as the substrate is oxidized .

• Oxygen consumption: Using Clark-type electrodes to measure oxygen uptake during enzyme activity.

• UV-spectrophotometer analysis: Can demonstrate the enzyme's ability to catalyze the oxidation of substrate cytochrome c .

How do molecular docking studies inform our understanding of inhibitor binding to MT-CO2?

Molecular docking studies provide crucial insights into the interaction between MT-CO2 and potential inhibitors or modulators, offering a structural basis for understanding enzyme regulation:

• Binding site identification: Docking studies can reveal potential binding pockets within the MT-CO2 structure where inhibitors may interact. For example, research has shown that allyl isothiocyanate (AITC) can interact with MT-CO2, influencing its catalytic activity .

• Interaction mapping: Molecular docking has identified specific residues involved in ligand binding. For instance, it was found that a sulfur atom in the AITC structure can form a hydrogen bond of 2.9 Å length with Leu-31 in MT-CO2 . This type of precise structural information helps understand the molecular basis of inhibition.

• Structure-activity relationships: By docking multiple inhibitor variants, researchers can correlate chemical structures with inhibitory potency, guiding rational inhibitor design.

• Conformational changes: Docking studies can predict how inhibitor binding might induce conformational changes in the protein that affect its function.

• Species-specific differences: Comparative docking using MT-CO2 models from different species helps identify conserved versus variable binding sites, explaining selectivity.

These computational approaches are particularly valuable when combined with experimental validation through site-directed mutagenesis, where predicted interaction residues are altered to confirm their importance in inhibitor binding.

What are the challenges in expressing soluble, functional recombinant MT-CO2, and how can they be addressed?

Expressing soluble and functional recombinant Pseudalopex sechurae MT-CO2 presents several challenges that researchers must overcome:

• Inclusion body formation: As a membrane-associated protein with hydrophobic regions, MT-CO2 often aggregates into inclusion bodies when overexpressed in E. coli.

  • Solution: Expression at lower temperatures (16-20°C), reduced inducer concentrations, and use of solubility-enhancing fusion partners like thioredoxin or MBP.

• Copper center formation: The functional CuA center requires proper incorporation of copper ions.

  • Solution: Supplementation of growth media with copper sulfate (5-10 μM CuSO₄) and careful oxidation conditions during protein refolding.

• Protein instability: Purified MT-CO2 may show reduced stability outside its native membrane environment.

  • Solution: Use of stabilizing additives (glycerol, trehalose) in buffer formulations and optimized storage conditions.

• Improper folding: Achieving the correct tertiary structure is essential for activity.

  • Solution: Co-expression with chaperone proteins (GroEL/GroES) and refolding protocols specifically designed for copper-containing proteins.

• Low expression yield: Mitochondrial proteins often express poorly in bacterial systems.

  • Solution: Codon optimization for E. coli and use of specialized expression strains like Transetta(DE3) that can enhance expression of proteins with rare codons .

Successful strategies have included using the pET-32a expression vector with an IPTG-inducible system in E. coli Transetta(DE3), followed by purification using affinity chromatography with Ni²⁺-NTA agarose, yielding approximately 50 μg/mL of functional protein .

How should researchers design comparative studies between MT-CO2 from Pseudalopex sechurae and other species?

Designing robust comparative studies between Pseudalopex sechurae MT-CO2 and orthologs from other species requires careful consideration of multiple parameters:

• Sequence alignment and phylogenetic analysis:

  • Conduct multiple sequence alignment to identify conserved and variable regions

  • Perform phylogenetic analysis to establish evolutionary relationships between species

  • Focus on copper-binding motifs and functional domains for targeted comparisons

  • Example approach: Multiple sequence alignment has indicated that Sitophilus zeamais COXII had high sequence identity with COXII of other insect species

• Structural comparison protocol:

  • Generate homology models based on available crystal structures

  • Compare secondary structure elements, particularly the transmembrane domains

  • Analyze conservation of the CuA center geometry

  • Map species-specific variations onto 3D structures to identify surface versus core differences

• Functional comparison design:

  • Express recombinant MT-CO2 from multiple species using identical expression and purification protocols

  • Standardize enzymatic assays to ensure comparable conditions

  • Measure kinetic parameters (Km, Vmax, kcat) under identical conditions

  • Compare substrate specificity and inhibitor sensitivity profiles

• Controls and normalization:

  • Include well-characterized reference species (e.g., human, bovine) in all analyses

  • Normalize activity data to copper content to account for differences in metal incorporation

  • Use internal standards for all quantitative measurements

  • Include technical and biological replicates to ensure statistical validity

This multi-faceted approach enables researchers to distinguish species-specific adaptations from conserved features, providing insights into the evolutionary and functional significance of variations in MT-CO2 across species.

What controls are essential when analyzing inhibitor effects on recombinant MT-CO2?

When studying the effects of inhibitors on recombinant Pseudalopex sechurae MT-CO2, including appropriate controls is critical for generating reliable and interpretable results:

• Activity baseline controls:

  • Untreated enzyme: Establish baseline activity without any inhibitor

  • Vehicle control: Include all solvents used for inhibitor solubilization

  • Time-matched controls: Account for any time-dependent decrease in enzyme activity

  • These controls ensure that observed effects are attributable to the inhibitor rather than experimental artifacts

• Inhibition specificity controls:

  • Known inhibitors: Include established inhibitors (e.g., cyanide, azide) as positive controls

  • Structurally related non-inhibitors: Compounds similar to the test inhibitor but lacking activity

  • Cross-validation with different activity assays: Confirm inhibition using multiple methodologies

  • For example, when studying AITC effects on MT-CO2, control experiments should include structurally similar compounds that don't affect enzyme activity

• Concentration-response controls:

  • Multiple inhibitor concentrations: Establish dose-response relationships

  • Sub-inhibitory concentrations: Verify threshold effects

  • Supra-maximal concentrations: Confirm complete inhibition plateaus

  • Reversibility test: Enzyme activity recovery after inhibitor removal

• Protein quality controls:

  • Multiple protein batches: Ensure reproducibility across preparations

  • Activity verification: Confirm protein is catalytically active before inhibitor studies

  • Proper folding verification: Spectroscopic confirmation of intact CuA center

• Experimental design controls:

  • Randomization: Randomize the order of sample processing

  • Blinding: When feasible, blind the identity of test compounds

  • Technical replicates: Minimum of triplicate measurements

  • Independent experimental repeats: At least three separate experiments

These comprehensive controls help distinguish specific inhibitor effects from artifacts and provide the foundation for mechanistic interpretations of inhibition data.

How can site-directed mutagenesis be applied to study the functional domains of MT-CO2?

Site-directed mutagenesis offers a powerful approach to dissect the structure-function relationships within Pseudalopex sechurae MT-CO2, particularly for understanding the roles of specific amino acids in copper binding and catalytic activity:

• Strategic mutation target selection:

  • Copper-binding residues: Conserved cysteines at positions 196 and 200, and histidine at position 204 that coordinate the CuA center

  • Substrate interaction sites: Residues predicted to interact with cytochrome c

  • Transmembrane anchoring domains: Amino acids in the N-terminal transmembrane helices

  • Potential inhibitor binding sites: Residues like Leu-31 identified in molecular docking studies with inhibitors like AITC

• Mutation design principles:

  • Conservative substitutions: Replace with chemically similar amino acids to test specific chemical properties

  • Alanine scanning: Systematic replacement with alanine to remove side-chain interactions

  • Charge reversal: Switch acidic to basic residues to test electrostatic interactions

  • Cross-species substitutions: Replace residues with those found in other species to test evolutionary adaptations

• Expression and purification strategy:

  • Express wild-type and mutant proteins in parallel using identical conditions

  • Use the same vector system (e.g., pET-32a) and expression host (e.g., E. coli Transetta DE3)

  • Purify using standardized protocols with affinity chromatography

  • Validate proper folding of mutants using spectroscopic techniques

• Functional characterization:

  • Enzymatic activity: Compare cytochrome c oxidation rates between wild-type and mutants

  • Copper content: Quantify copper incorporation using atomic absorption or ICP-MS

  • Thermal stability: Assess structural integrity using differential scanning fluorimetry

  • Inhibitor sensitivity: Compare effects of inhibitors on wild-type versus mutant proteins

• Data interpretation framework:

  • Correlation matrix: Relate structural changes to functional effects

  • Additivity analysis: Test whether multiple mutations have additive or synergistic effects

  • Structure mapping: Project results onto structural models to visualize functional domains

This systematic mutagenesis approach allows researchers to build a comprehensive map of structure-function relationships in MT-CO2, identifying residues critical for copper binding, electron transfer, protein stability, and inhibitor interactions.

What purification strategies yield the highest purity and activity for recombinant MT-CO2?

Optimizing purification of recombinant Pseudalopex sechurae MT-CO2 requires specific strategies to maintain protein integrity while achieving high purity:

• Affinity chromatography optimization:

  • Nickel-based affinity chromatography (Ni²⁺-NTA agarose) is the primary method for His-tagged MT-CO2 purification

  • Imidazole gradient elution (20-250 mM) provides better separation than step elution

  • Slow flow rates (0.5-1 ml/min) improve binding efficiency and reduce column pressure

  • Addition of low concentrations of detergents (0.05-0.1% Triton X-100) can improve solubility

• Buffer composition considerations:

  • Maintain pH between 7.2-7.5 using phosphate or Tris-based buffers

  • Include 150-300 mM NaCl to reduce non-specific interactions

  • Add glycerol (5-10%) as a stabilizing agent

  • Supplement with copper ions (5-10 μM CuSO₄) to support CuA center assembly

  • Include reducing agents (0.5-1 mM DTT) to prevent oxidation of cysteine residues

• Multi-step purification approach:

  • Initial IMAC (Immobilized Metal Affinity Chromatography) captures the His-tagged protein

  • Size exclusion chromatography as a polishing step separates aggregates and oligomers

  • Ion exchange chromatography can remove contaminants with different charge properties

• Quality assessment criteria:

  • SDS-PAGE with Coomassie staining should show >90% purity

  • Western blotting using anti-His antibodies confirms identity

  • Copper content analysis verifies metal incorporation

  • Activity assays validate functional integrity

The optimized protocol typically yields protein with specific activity comparable to that of native enzyme, with preserved copper centers and catalytic function .

What spectroscopic methods are most informative for characterizing the copper center in MT-CO2?

Spectroscopic characterization of the CuA center in recombinant Pseudalopex sechurae MT-CO2 provides critical insights into its structural integrity and functional state:

• UV-Visible Spectroscopy:

  • The CuA center exhibits characteristic absorption bands at 480-500 nm and 530-550 nm

  • Oxidized and reduced states show distinct spectral signatures

  • Monitoring these spectra can confirm proper formation of the copper center

  • This method has been successfully used to show that recombinant COXII can catalyze the oxidation of substrate Cytochrome C

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Detects unpaired electrons in the CuA center

  • Oxidized CuA center gives a characteristic EPR signal with g-values around 2.0

  • Provides information about the electronic structure and coordination environment

  • Temperature-dependent measurements (typically at liquid nitrogen temperatures) enhance signal quality

• X-ray Absorption Spectroscopy (XAS):

  • X-ray Absorption Near Edge Structure (XANES) determines copper oxidation states

  • Extended X-ray Absorption Fine Structure (EXAFS) reveals bond distances and coordination geometry

  • Non-destructive method that works with proteins in solution

• Resonance Raman Spectroscopy:

  • Identifies vibrational modes of the Cu-S bonds in the CuA center

  • Excitation wavelengths of 600-700 nm enhance copper center signals

  • Distinguishes Cu-S stretching modes (~270-290 cm⁻¹) from other metal-ligand vibrations

• Infrared Spectroscopy:

  • Can detect structural changes in the protein upon interaction with substrates or inhibitors

  • Has been used to analyze how compounds like AITC influence the catalytic activity of COXII

These complementary spectroscopic techniques provide a comprehensive characterization of the copper center, confirming proper metal incorporation and electronic structure, which are prerequisites for functional activity in recombinant MT-CO2.

What are the optimal storage conditions for maintaining activity of purified recombinant MT-CO2?

Preserving the stability and activity of recombinant Pseudalopex sechurae MT-CO2 requires careful attention to storage conditions:

• Short-term storage (1-7 days):

  • Temperature: 4°C

  • Buffer composition: 20-50 mM phosphate buffer or Tris-HCl, pH 7.4, with 150 mM NaCl

  • Additives: 5-10% glycerol as a stabilizer

  • Storage container: Low protein-binding tubes

  • Expected stability: >90% activity retention

• Medium-term storage (1-3 months):

  • Temperature: -20°C with cryoprotectants

  • Additives: 10-15% glycerol and 1-2% sucrose or trehalose

  • Aliquoting: Divide into single-use volumes to avoid freeze-thaw cycles

  • Expected stability: 70-80% activity retention

• Long-term storage (>3 months):

  • Primary method: Lyophilization (freeze-drying)

  • Pre-lyophilization treatment: Addition of stabilizers (trehalose, sucrose)

  • Storage of lyophilized material: -80°C with desiccant

  • Expected stability: >60% activity for 6-12 months when properly lyophilized

• Stability monitoring protocol:

  • Periodic activity testing using standardized cytochrome c oxidation assay

  • Spectroscopic analysis to monitor copper center integrity

  • SDS-PAGE to assess degradation

• Reconstitution of lyophilized protein:

  • Slow addition of ice-cold buffer with gentle mixing

  • Allow complete dissolution before use

  • Centrifugation step to remove any insoluble material

  • Activity verification before experimental use

Optimal storage conditions vary depending on the specific application requirements and desired storage duration. For critical applications requiring maximum activity, freshly purified protein or short-term storage at 4°C is recommended, while lyophilization offers the best option for long-term preservation of stability .

How can recombinant MT-CO2 be used in evolutionary biology studies?

Recombinant Pseudalopex sechurae MT-CO2 offers valuable opportunities for evolutionary biology investigations:

• Phylogenetic analysis applications:

  • Sequence comparison of MT-CO2 across canid species provides insights into evolutionary relationships

  • Multiple sequence alignment and phylogenetic analysis have shown high sequence identity between MT-CO2 proteins from different species

  • Constructing phylogenetic trees based on MT-CO2 sequences can help resolve taxonomic relationships within the Canidae family

  • The 227-amino acid sequence of Pseudalopex sechurae MT-CO2 can be compared with other species to identify conserved and variable regions

• Functional evolution studies:

  • Comparing enzymatic properties of recombinant MT-CO2 from Pseudalopex sechurae with other species reveals functional adaptations

  • Kinetic parameters (Km, Vmax) may reflect ecological adaptations to different environments

  • Thermal stability profiles can provide insights into adaptation to different temperature regimes

  • Inhibitor sensitivity differences may indicate species-specific regulatory mechanisms

• Molecular adaptation analysis:

  • Calculating selection pressures (dN/dS ratios) on different regions of the MT-CO2 gene identifies sites under positive or purifying selection

  • Mapping sequence variations onto 3D structure models reveals functionally significant adaptations

  • Correlating sequence changes with habitat differences can identify environmental adaptations

  • Experimental validation of adaptive hypotheses using site-directed mutagenesis

• Copper binding site evolution:

  • The conserved nature of the CuA center residues (cysteines at positions 196 and 200, histidine at 204) reflects fundamental functional constraints

  • Subtle variations in surrounding residues may influence redox properties and electron transfer efficiency

  • Comparing these features across species provides insights into the evolution of electron transport systems

This research direction contributes to our understanding of how mitochondrial proteins evolve under different selective pressures and how functional constraints shape molecular evolution patterns in metabolically critical proteins.

What are the potential applications of recombinant MT-CO2 in developing research tools?

Recombinant Pseudalopex sechurae MT-CO2 can be developed into various research tools with applications across multiple disciplines:

• Antibody production and validation:

  • Purified recombinant MT-CO2 serves as an excellent antigen for generating specific antibodies

  • These antibodies can be used for:

    • Western blot detection of MT-CO2 in tissue samples

    • Immunohistochemistry to visualize mitochondrial distribution

    • Immunoprecipitation for protein interaction studies

    • Flow cytometry to assess mitochondrial content in cells

• Enzymatic assay development:

  • Recombinant MT-CO2 can be incorporated into standardized assays for:

    • Screening inhibitors of mitochondrial respiration

    • Testing environmental toxicants that affect respiratory complex function

    • Evaluating compounds that might protect against mitochondrial dysfunction

    • The established cytochrome c oxidation assay provides a foundation for these applications

• Protein interaction studies:

  • His-tagged recombinant MT-CO2 enables identification of:

    • Protein partners in the respiratory chain

    • Regulatory factors that modulate activity

    • Species-specific interaction differences

    • Techniques like pull-down assays, surface plasmon resonance, and cross-linking mass spectrometry can leverage the purified protein

• Structural biology resources:

  • Well-characterized recombinant MT-CO2 can accelerate:

    • X-ray crystallography studies of the copper center

    • Cryo-EM analysis of respiratory complexes

    • NMR investigations of protein dynamics

    • Computational modeling validation

• Educational and training applications:

  • Purified recombinant MT-CO2 provides excellent material for:

    • Laboratory courses on protein purification

    • Enzyme kinetics practical training

    • Spectroscopic methods demonstration

    • Biophysical characterization workshops

These diverse applications highlight how a well-characterized recombinant protein can serve as a versatile resource for both basic and applied research across biochemistry, molecular biology, and biotechnology fields.