Recombinant Meriones shawi Cytochrome c oxidase subunit 2 (MT-CO2)

What is Meriones shawi Cytochrome c oxidase subunit 2 (MT-CO2)?

Meriones shawi Cytochrome c oxidase subunit 2 (MT-CO2) is a mitochondrial protein that functions as a core component of the cytochrome c oxidase (COX) complex, which serves as the terminal enzyme in the mitochondrial electron transport chain. The full-length protein consists of 228 amino acids and plays a crucial role in cellular respiration by facilitating electron transfer from cytochrome c to molecular oxygen, contributing to the generation of the proton gradient essential for ATP production .

MT-CO2 contains a dual core CuA active site that is directly responsible for the initial acceptance of electrons from cytochrome c. This process is fundamental to aerobic respiration and energy production in cells. The protein is encoded by the mitochondrial genome (MT-CO2 gene) of Meriones shawi (Shaw's jird), a rodent species . The recombinant version is typically expressed with an N-terminal His-tag to facilitate purification and experimental manipulation.

How does MT-CO2 function in electron transport and cellular respiration?

MT-CO2 functions as a critical electron transfer component within the cytochrome c oxidase complex. The process begins when reduced cytochrome c binds to the CuA center located in MT-CO2. From this initial binding site, electrons flow from cytochrome c to the CuA center, which consists of two copper atoms bridged by cysteine residues . This electron transfer represents the first step in a series of redox reactions culminating in the reduction of molecular oxygen to water.

The electron transfer pathway proceeds as follows:

• Cytochrome c → CuA center in MT-CO2

• CuA center → heme a

• Heme a → binuclear center (heme a3-CuB)

• Binuclear center → O₂ reduction to H₂O

This electron movement is coupled with proton pumping across the inner mitochondrial membrane, generating an electrochemical gradient that drives ATP synthesis. MT-CO2's position at the beginning of this electron transfer chain makes it essential for efficient oxidative phosphorylation . Studies have shown that structural changes in the MT-CO2 protein, particularly around the CuA site, can significantly affect the rate and efficiency of electron transfer, highlighting its central role in cellular bioenergetics .

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

Based on current research, Escherichia coli represents the most commonly utilized expression system for recombinant Meriones shawi MT-CO2 production. Several E. coli strains have demonstrated successful expression, including the Transetta (DE3) system . When designing an expression strategy, researchers should consider the following methodological approaches:

• Vector selection:

  • pET series vectors (particularly pET-32a) have been successfully used for MT-CO2 expression

  • Vectors with tightly controlled promoters help manage potential toxicity

• Expression conditions:

  • Induction with IPTG (isopropyl β-d-thiogalactopyranoside) at concentrations between 0.1-1.0 mM

  • Lower induction temperatures (16-25°C) typically yield better results than standard 37°C expression

  • Extended expression times (16-24 hours) at lower temperatures improve proper folding

• Fusion partners and tags:

  • N-terminal His-tag facilitates purification via nickel affinity chromatography

  • MBP (maltose-binding protein) fusion can enhance solubility

  • Tag placement should consider the protein's functional domains to minimize interference

Alternative expression systems worth considering include:

• Insect cell systems (Sf9, Sf21) for improved post-translational modifications

• Yeast expression systems for membrane-associated proteins

• Cell-free expression systems for rapid screening or when conventional systems fail

Expression optimization should be systematically approached through small-scale experiments varying temperature, inducer concentration, and incubation time before scaling up to preparative quantities .

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

Purifying recombinant MT-CO2 while maintaining its structural integrity and functional activity requires careful consideration of multiple factors. The following multi-step purification approach has proven effective:

• Initial capture using affinity chromatography:

  • Ni²⁺-NTA agarose chromatography for His-tagged protein

  • Gradual imidazole gradient (20-250 mM) minimizes non-specific binding

  • Inclusion of glycerol (5-10%) in buffers enhances protein stability during purification

• Secondary purification steps:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Ion exchange chromatography to remove remaining impurities

  • Careful buffer optimization to maintain the CuA center integrity

• Critical buffer components:

  • Tris/PBS-based buffers at pH 7.5-8.0 maintain stability

  • Addition of 6% trehalose prevents aggregation during concentration

  • Copper supplementation (10-50 μM CuSO₄) may enhance CuA center formation

For maximum yield and purity, researchers should implement the following protocol:

• Lyse cells in buffer containing protease inhibitors and DNase I

• Clarify lysate by high-speed centrifugation (20,000 × g, 30 min)

• Apply supernatant to pre-equilibrated Ni²⁺-NTA column

• Wash with increasing imidazole concentrations

• Elute with high imidazole (250-300 mM)

• Perform buffer exchange to remove imidazole

• Concentrate to 0.1-1.0 mg/mL

Final purified protein should be assessed by SDS-PAGE (>90% purity) and Western blotting with anti-His antibodies to confirm identity. Functional assays examining electron transfer capability should be performed to verify activity preservation throughout the purification process .

How can researchers assess the enzymatic activity of recombinant MT-CO2?

Assessing the enzymatic activity of recombinant MT-CO2 requires specialized techniques that measure electron transfer capability. Several complementary methodological approaches can be employed:

• Spectrophotometric assays:

  • Cytochrome c oxidation can be monitored at 550 nm, tracking the decrease in absorbance as reduced cytochrome c is oxidized

  • Reaction mixture typically contains 10-50 μM reduced cytochrome c, purified MT-CO2, and appropriate buffer (pH 7.0-7.5)

  • Activity is calculated as the rate of absorbance change (ΔA₅₅₀/min) normalized to protein concentration

• Polarographic oxygen consumption measurements:

  • Clark-type oxygen electrode measures oxygen uptake rates

  • Standard reaction contains reduced cytochrome c, MT-CO2, and air-saturated buffer

  • Activity is reported as nmol O₂ consumed/min/mg protein

• Advanced spectroscopic analysis:

  • UV-visible spectroscopy can detect characteristic absorption peaks of the CuA center

  • Resonance Raman spectroscopy reveals structural changes around metal centers during catalysis

  • Infrared spectroscopy can be used to analyze the impact of substrates or inhibitors on protein structure

• Binding affinity determination:

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure interaction with cytochrome c

  • Determine binding constants (Kd) and thermodynamic parameters of interaction

A practical approach involves establishing a standardized activity assay protocol where activity is compared to a well-characterized reference standard (such as bovine heart cytochrome c oxidase). This allows for meaningful comparison between different preparation methods and experimental conditions .

What factors affect MT-CO2 catalytic activity and how can they be controlled in experimental settings?

Multiple factors influence the catalytic activity of MT-CO2, and controlling these variables is essential for experimental reproducibility and meaningful data interpretation:

• pH dependency:

  • MT-CO2 activity typically exhibits a bell-shaped pH profile with optimum between pH 7.0-7.5

  • Activity decreases sharply below pH 6.5 or above pH 8.0

  • Buffer systems should maintain constant pH throughout experiments (HEPES or phosphate buffers are commonly used)

• Temperature effects:

  • Activity increases with temperature up to an optimum (typically 30-37°C for mammalian proteins)

  • Thermal stability tests should be conducted to determine the temperature range for stable activity

  • Temperature control within ±0.5°C is recommended during kinetic measurements

• Metal ion requirements:

  • The CuA center requires proper copper incorporation for activity

  • Trace amounts of other metal ions (particularly iron) may influence activity

  • Metal chelators should be avoided in experimental buffers

• Redox environment:

  • The oxidation state of the CuA center affects its ability to accept electrons

  • Mild reducing agents may be necessary to maintain the protein in the proper redox state

  • Oxygen concentration should be controlled when measuring activity under different conditions

• Regulatory interactions:

  • Proteins like Higd1a can significantly enhance cytochrome c oxidase activity by causing structural changes around the active site

  • These interactions can increase activity up to twofold under experimental conditions

  • Small molecules such as allyl isothiocyanate (AITC) can influence activity by binding to specific sites on the protein

• Storage conditions impact:

  • Freeze-thaw cycles significantly reduce activity

  • Protein should be stored at -20°C/-80°C in aliquots to prevent repeated freezing

  • Addition of 6% trehalose and storage in Tris/PBS-based buffer preserves activity

To systematically control these factors, researchers should:

• Establish standard assay conditions with carefully controlled pH, temperature, and ionic strength

• Include appropriate controls in each experiment

• Prepare fresh substrate solutions for each set of measurements

• Validate activity periodically against reference standards

What techniques are most effective for analyzing the structure-function relationship of MT-CO2?

Understanding the structure-function relationship of MT-CO2 requires a multi-technique approach that examines both static structure and dynamic changes during catalysis:

• Spectroscopic techniques:

  • Resonance Raman spectroscopy provides detailed information about the environment around the heme groups and metal centers

  • Changes in specific vibrational bands correlate with structural rearrangements during electron transfer

  • This technique has revealed that proteins like Higd1a cause structural changes around heme a, influencing electron transfer rates

• X-ray crystallography and cryo-EM:

  • These techniques provide atomic-resolution structures, though typically requiring incorporation into the complete COX complex

  • Structural data reveals the precise coordination geometry of the CuA center and its relationship to other functional domains

  • Comparison of structures in different redox states can identify conformational changes essential for function

• Molecular dynamics simulations:

  • Computational modeling complements experimental data by predicting protein dynamics

  • Simulations can identify transient states difficult to capture experimentally

  • Water molecule and proton movement pathways can be mapped

• Site-directed mutagenesis combined with activity assays:

  • Systematic mutation of key residues (particularly those coordinating the CuA center)

  • Structure-function correlations established by measuring activity changes

  • Conservation analysis across species identifies functionally critical residues

• Molecular docking studies:

  • Computational approaches can predict binding interactions with substrates or regulators

  • For instance, molecular docking has shown that allyl isothiocyanate (AITC) can form a 2.9 Å hydrogen bond with Leu-31 in COXII, potentially explaining its regulatory effect

The most effective approach integrates multiple techniques to build a comprehensive understanding of how MT-CO2 structure relates to its function in electron transfer. This integrated approach has revealed that subtle structural changes around the metal centers can significantly impact catalytic efficiency, explaining how regulatory proteins like Higd1a can enhance activity .

How does the CuA active site in MT-CO2 contribute to electron transfer mechanisms?

The CuA active site in MT-CO2 serves as the primary electron acceptor from cytochrome c and exhibits unique structural and electronic properties that facilitate efficient electron transfer:

• CuA center structure:

  • Contains a binuclear copper center with two copper atoms bridged by two cysteine thiolate ligands

  • Additional coordination by histidine, methionine, and backbone carbonyl ligands

  • This creates a unique purple-colored center with distinctive spectroscopic properties

• Electronic properties facilitating electron transfer:

  • The binuclear nature of CuA allows for electron delocalization between the two copper atoms

  • This creates a mixed-valence state (Cu⁺-Cu²⁺) that stabilizes the one-electron reduced form

  • The delocalized electronic structure enhances electron tunneling rates by providing a more favorable electronic coupling pathway

• Redox properties:

  • The CuA center has a redox potential of approximately +240 mV (vs. standard hydrogen electrode)

  • This potential is carefully tuned to be lower than cytochrome c (+254 mV) but higher than heme a (+210 mV)

  • The optimized potential gradient facilitates directional electron flow through the enzyme

• Structural adaptations for interaction with cytochrome c:

  • The surface region surrounding CuA contains negatively charged residues that complement positively charged residues on cytochrome c

  • This electrostatic steering optimizes the orientation for efficient electron transfer

  • Molecular docking studies and mutagenesis experiments have identified specific interaction sites

• Conformational changes during electron transfer:

  • Electron acceptance by the CuA center triggers subtle structural changes

  • These changes propagate through the protein and facilitate subsequent electron transfer to heme a

  • Studies using resonance Raman spectroscopy have shown that regulatory proteins like Higd1a can influence these structural transitions, altering the rate of electron transfer

How does Meriones shawi MT-CO2 compare structurally and functionally to MT-CO2 from other species?

Comparative analysis of Meriones shawi MT-CO2 with orthologs from other species reveals important insights about structural conservation, functional adaptations, and evolutionary relationships:

• Sequence conservation analysis:

This comparative approach not only enhances our understanding of MT-CO2 evolution but also provides insights into how protein structure and function can be maintained despite substantial sequence variation.

What insights into mitonuclear coevolution can be gained from studying MT-CO2?

MT-CO2 provides an excellent model for studying mitonuclear coevolution due to its mitochondrial encoding and essential interactions with nuclear-encoded proteins:

• Mitonuclear protein interactions:

  • MT-CO2 must maintain functional interactions with nuclear-encoded subunits of cytochrome c oxidase

  • The CuA center in MT-CO2 accepts electrons from nuclear-encoded cytochrome c

  • These interactions create selective pressure for coordinated evolution between mitochondrial and nuclear genomes

• Evidence from population studies:

  • Research on Tigriopus californicus has revealed that approximately 4% of sites in the COII gene appear to evolve under relaxed selective constraint (ω = 1), while the majority of codons show strong purifying selection (ω << 1)

  • A branch-site maximum likelihood model identified three sites that may have experienced positive selection within specific population clades

  • These patterns suggest adaptive evolution to maintain compatibility with nuclear-encoded partners

• Hybrid incompatibility mechanisms:

  • Studies have shown functional and fitness consequences in interpopulation hybrids between central and northern California populations of T. californicus

  • These incompatibilities likely result from mismatches between co-adapted mitochondrial and nuclear genes

  • MT-CO2 mutations may require compensatory changes in nuclear-encoded interacting partners

• Implications for speciation:

  • Mitonuclear incompatibilities involving MT-CO2 and its interaction partners could contribute to reproductive isolation

  • The rapid evolution of MT-CO2 in some lineages might accelerate this process

  • Understanding these dynamics provides insights into mechanisms of speciation

• Methodological approaches for studying mitonuclear coevolution:

  • Comparative genomics between populations with different MT-CO2 sequences

  • Functional assays measuring electron transfer efficiency in hybrid systems

  • Reconstruction of ancestral sequences to trace evolutionary trajectories

  • Creation of chimeric proteins to identify regions critical for compatibility

• Applications to human disease:

  • Insights from natural variation in MT-CO2 could help interpret the pathogenicity of human MT-CO2 mutations

  • Understanding compensatory mechanisms might suggest therapeutic approaches for mitochondrial disorders

  • Natural variants with enhanced function could inspire protein engineering for disease treatment

Studying mitonuclear coevolution through the lens of MT-CO2 not only advances evolutionary biology but also has practical implications for understanding and treating mitochondrial disorders.

How can recombinant MT-CO2 be utilized in bioenergetics and mitochondrial research?

Recombinant Meriones shawi MT-CO2 offers several valuable applications in bioenergetics and mitochondrial research:

• Structure-function relationship studies:

  • Site-directed mutagenesis of key residues to analyze their role in electron transfer

  • Creation of chimeric proteins combining domains from different species

  • In vitro reconstitution with other cytochrome c oxidase subunits to study assembly and function

• Electron transfer mechanism investigation:

  • Direct measurement of electron transfer rates between cytochrome c and MT-CO2

  • Analysis of factors affecting electron tunneling efficiency

  • Examination of how protein dynamics influence electron transfer

• Inhibitor and modulator screening:

  • Identification of novel compounds that affect MT-CO2 function

  • Characterization of binding sites using techniques like molecular docking

  • For example, studies have shown that allyl isothiocyanate (AITC) can influence MT-CO2 activity by forming specific hydrogen bonds with the protein

• Regulation of cytochrome c oxidase activity:

  • Investigation of how proteins like Higd1a enhance cytochrome c oxidase activity

  • Studies have demonstrated that regulatory proteins can cause structural changes around heme groups, effectively doubling enzyme activity under certain conditions

  • Analysis of post-translational modifications affecting MT-CO2 function

• Biophysical technique development:

  • Serving as a model system for developing new methods to study membrane proteins

  • Providing purified protein standards for assay development

  • Testing novel spectroscopic approaches for analyzing metalloproteins

• Evolutionary and comparative studies:

  • Comparing properties of MT-CO2 from different species to understand adaptive variations

  • Analysis of sequence-function relationships across evolutionary distance

  • Investigation of mitonuclear co-evolution through MT-CO2 interactions with nuclear-encoded proteins

• Metabolic engineering applications:

  • Enhancing respiratory chain efficiency through engineered MT-CO2 variants

  • Development of MT-CO2-based biosensors for metabolic studies

  • Creation of minimal respiratory systems for synthetic biology applications

These research applications rely on the ability to produce pure, functional recombinant MT-CO2, underscoring the importance of optimized expression and purification protocols. The resulting insights contribute significantly to our understanding of mitochondrial function and energy metabolism.

What protocols are most effective for studying MT-CO2 interactions with regulatory proteins?

Investigating interactions between MT-CO2 and regulatory proteins requires carefully designed protocols that preserve protein structure and function while providing quantitative interaction data:

• Protein-protein interaction detection methods:

  • Co-immunoprecipitation (Co-IP) using antibodies against MT-CO2 or the regulatory protein

  • Pull-down assays utilizing recombinant tagged proteins

  • Crosslinking coupled with mass spectrometry to identify interaction interfaces

  • Surface plasmon resonance (SPR) for real-time kinetic analysis of binding

• Functional impact assessment:

  • Activity assays before and after addition of regulatory proteins

  • For example, direct addition of MBP-Higd1a to highly purified cytochrome c oxidase has been shown to significantly increase enzyme activity to twice that of the enzyme alone

  • Dose-response experiments to determine EC₅₀ values

  • Competition assays to investigate binding specificity

• Structural characterization of complexes:

  • Resonance Raman spectroscopy to detect structural changes in MT-CO2 upon regulator binding

  • This approach revealed that Higd1a causes structural changes around heme a within the cytochrome c oxidase complex

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

  • Single-particle cryo-EM for visualizing the complete complex architecture

• Interaction mapping protocol:

  • Generate truncation mutants of both MT-CO2 and the regulatory protein

  • Perform systematic binding assays to identify minimal interaction domains

  • Follow with site-directed mutagenesis of key residues

  • Validate findings using structural and functional assays

• Computational approaches:

  • Molecular docking to predict binding interfaces

  • Molecular dynamics simulations to analyze stability of complexes

  • This approach has been successfully applied to study the interaction between AITC and COXII, identifying a specific hydrogen bond with Leu-31

• In-cell validation:

  • Proximity ligation assays to confirm interactions in a cellular context

  • FRET-based approaches to visualize interactions in living cells

  • Genetic approaches (e.g., suppressor screens) to identify functional relationships

A comprehensive protocol would integrate multiple approaches:

• Initial screening using pull-down or Co-IP to confirm interaction

• Quantitative binding analysis using SPR or ITC

• Functional impact assessment through activity assays

• Structural characterization using spectroscopic methods

• Detailed mapping of interaction interface

• Validation in cellular context

This multi-faceted approach provides robust evidence for physiologically relevant interactions and their functional significance.

What are the most common challenges in expressing and purifying functional MT-CO2 and how can they be addressed?

Researchers working with recombinant MT-CO2 frequently encounter several technical challenges that can be systematically addressed with appropriate strategies:

• Low expression yields:

  • Challenge: MT-CO2 often expresses poorly in heterologous systems

  • Solutions:

    • Optimize codon usage for the expression host

    • Lower induction temperature (16-20°C) to slow expression rate

    • Try different E. coli strains specifically designed for membrane proteins

    • Consider fusion partners known to enhance expression (MBP, SUMO, Trx)

• Inclusion body formation:

  • Challenge: MT-CO2 tends to form insoluble aggregates in E. coli

  • Solutions:

    • Reduce inducer concentration (0.1-0.2 mM IPTG)

    • Co-express with molecular chaperones (GroEL/GroES)

    • Add mild detergents or glycerol to lysis buffer

    • Develop refolding protocols if soluble expression fails

• Improper cofactor incorporation:

  • Challenge: The CuA site may not form correctly in recombinant protein

  • Solutions:

    • Supplement growth media with copper (50-100 μM CuSO₄)

    • Co-express with copper chaperones

    • Consider in vitro reconstitution of the CuA site after purification

    • Verify copper incorporation using UV-visible spectroscopy

• Protein instability during purification:

  • Challenge: MT-CO2 may denature or aggregate during purification

  • Solutions:

    • Include stabilizing agents (glycerol, trehalose) in all buffers

    • Perform all purification steps at 4°C

    • Minimize exposure to air/oxidation

    • Work quickly and avoid unnecessary freeze-thaw cycles

• Low enzymatic activity:

  • Challenge: Purified protein shows poor electron transfer activity

  • Solutions:

    • Verify structural integrity using spectroscopic methods

    • Test activity enhancement with known activators like Higd1a

    • Consider reconstitution with other subunits of cytochrome c oxidase

    • Optimize buffer conditions (pH, ionic strength) for activity assays

• Aggregation during storage:

  • Challenge: Purified MT-CO2 aggregates during storage

  • Solutions:

    • Store in buffer containing 6% trehalose and 50% glycerol

    • Aliquot to avoid repeated freeze-thaw cycles

    • Store at -20°C/-80°C for long-term stability

    • Consider lyophilization for extended storage

A systematic troubleshooting approach recording expression, purification, and storage conditions alongside activity measurements will help identify optimal protocols for specific experimental needs. Learning from previous studies with similar proteins, like the successful expression of S. zeamais COXII in E. coli using pET-32a vectors and purification via Ni²⁺-NTA affinity chromatography, provides valuable starting points .

How can researchers verify the structural integrity and functional activity of purified recombinant MT-CO2?

Verifying both structural integrity and functional activity of purified recombinant MT-CO2 is essential for ensuring reliable experimental results. A comprehensive validation approach includes:

• Protein purity and identity verification:

  • SDS-PAGE analysis: Should show >90% purity with correct molecular weight (~26-27 kDa plus tag)

  • Western blotting: Using antibodies against the protein or tag to confirm identity

  • Mass spectrometry: For accurate mass determination and sequence verification

  • N-terminal sequencing: To confirm the start of the protein sequence

• Structural integrity assessment:

  • UV-visible spectroscopy: The CuA center has characteristic absorption features

  • Circular dichroism (CD): To assess secondary structure content and proper folding

  • Thermal shift assays: To determine protein stability

  • Dynamic light scattering (DLS): To check for aggregation and homogeneity

• Functional activity validation:

  • Cytochrome c oxidation assay: Monitoring the decrease in absorbance at 550 nm as reduced cytochrome c is oxidized

  • Oxygen consumption measurements: Using oxygen electrodes to measure the rate of oxygen reduction

  • Electron transfer rate determination: Using stopped-flow techniques to measure rapid kinetics

  • Response to known inhibitors and activators: Testing sensitivity to compounds like cyanide (inhibitor) or Higd1a (activator)

• Metal content analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS): To quantify copper content

  • EPR spectroscopy: To examine the redox state of the CuA center

  • Atomic absorption spectroscopy: For copper quantification

• Interaction with binding partners:

  • Surface plasmon resonance: To measure binding kinetics with cytochrome c

  • Isothermal titration calorimetry: For thermodynamic characterization of interactions

  • Co-immunoprecipitation: To verify interactions with known binding partners

• Comparative analysis:

  • Activity comparison with native enzyme: Benchmark against naturally occurring MT-CO2

  • Cross-species comparison: Test activity patterns against well-characterized orthologs

A practical validation protocol should include:

• Initial purity check by SDS-PAGE (>90% purity target)

• Western blot confirmation of identity

• UV-visible spectrum to verify CuA center integrity

• Basic activity assay measuring cytochrome c oxidation

• Stability assessment through thermal shift assay or activity retention over time

These validation steps should be documented with each protein preparation to ensure consistency between experiments and reliable interpretation of results .