Recombinant Mandrillus leucophaeus Cytochrome c oxidase subunit 2 (MT-CO2)

What is Mandrillus leucophaeus Cytochrome c oxidase subunit 2?

Mandrillus leucophaeus Cytochrome c oxidase subunit 2 (MT-CO2) is a mitochondrial protein component of the cytochrome c oxidase complex, which plays a crucial role in the electron transport chain and cellular energy production. This protein is encoded by the mitochondrial genome (mtDNA) of the Drill monkey (Mandrillus leucophaeus), a large-bodied primate native to regions in Cameroon, Nigeria, and Equatorial Guinea . MT-CO2 consists of 227 amino acids with the following sequence: MAHPAQLGLQDATSPVMEELITFHDHALMAMSLISLLVLYALFSTLTTKMTNTNITDAQEMETIWTILPAIILVLIAFPSLRILYMTDEVNNPSFTIKSIGHQWYWTYEYTDYGGLIFNSYMLPPLFLNPGDLRLLEVDNRVVLPIEAPVRMMITSQDVLHSWTIPTLGLKTDAVPGRLNQTVFTATRPGVYYGQCSEICGANHSFMPIVAELIPLKIFEMGPVFTL .

The protein functions as part of Complex IV in the respiratory chain, catalyzing the transfer of electrons from cytochrome c to molecular oxygen. This reaction is coupled with proton pumping across the inner mitochondrial membrane, contributing to the electrochemical gradient that drives ATP synthesis. Structurally, MT-CO2 contains transmembrane domains that anchor it within the inner mitochondrial membrane.

Why study MT-CO2 from Drill (Mandrillus leucophaeus)?

Studying MT-CO2 from Drill monkeys offers several significant research advantages. First, as a threatened primate species with a restricted geographic distribution, Drills represent an important evolutionary branch for comparative studies of mitochondrial function across primates . The Drill population is geographically isolated, with discontinuous distribution across at least 11 mainland areas and two populations on Bioko Island . This isolation may have led to unique genetic adaptations in essential proteins like MT-CO2.

Second, mitochondrial genes like MT-CO2 evolve at different rates compared to nuclear genes, making them valuable molecular markers for phylogenetic studies and understanding primate evolution. The study of MT-CO2 can provide insights into how energy metabolism has evolved across primate lineages and adapted to different ecological niches.

Third, comparing MT-CO2 structure and function between Drills and humans can illuminate the molecular basis of cytochrome c oxidase deficiencies, which cause severe metabolic disorders in humans . Since mitochondrial function is highly conserved yet shows species-specific variations, research on Drill MT-CO2 can highlight critical functional domains and potentially identify novel therapeutic approaches.

How is recombinant MT-CO2 produced?

Production of recombinant Mandrillus leucophaeus MT-CO2 typically follows these methodological steps:

• Gene synthesis and codon optimization: The MT-CO2 gene sequence (based on GenBank accession data) is synthesized with codon optimization for the selected expression system.

• Expression vector construction: The synthesized gene is cloned into an appropriate expression vector with a suitable promoter and affinity tag. Various tag types may be used depending on downstream applications and purification strategies .

• Host selection: Common expression systems include bacterial (E. coli), yeast (P. pastoris), insect cells (using baculovirus), or mammalian cells. For mitochondrial membrane proteins like MT-CO2, eukaryotic expression systems often yield better functional protein.

• Optimization of expression conditions: Parameters such as temperature, induction timing, and duration are optimized to maximize protein yield while maintaining proper folding.

• Protein extraction and purification: Since MT-CO2 is a membrane protein, specialized detergent-based extraction protocols are required. Purification typically involves affinity chromatography based on the incorporated tag, followed by size exclusion chromatography.

• Quality control: Purified protein is analyzed by SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity. Functional assays are performed to verify enzyme activity.

• Storage: The purified protein is stored in an optimized buffer containing 50% glycerol at -20°C or -80°C for extended storage .

This process yields recombinant protein suitable for various research applications, though challenges related to membrane protein expression and maintaining native conformation must be addressed.

What is the sequence conservation of MT-CO2 across primates?

MT-CO2 shows varying degrees of sequence conservation across primate species, reflecting both functional constraints and evolutionary divergence. The protein's core catalytic domains demonstrate higher conservation than peripheral regions, consistent with its essential role in cellular respiration.

Table 1: Sequence Identity Matrix of MT-CO2 Across Selected Primate Species

The high sequence similarity (96.5%) between Mandrillus leucophaeus and Mandrillus sphinx reflects their close evolutionary relationship, while comparison with humans (87.2% identity) reveals sufficient divergence to potentially explain species-specific differences in cytochrome c oxidase function. These sequence variations can be mapped to structural models to identify functionally significant adaptations across primate lineages.

How does Drill MT-CO2 compare functionally to human MT-CO2?

Functional comparison of Drill and human MT-CO2 reveals important differences in enzyme kinetics, stability, and interaction with other subunits of the cytochrome c oxidase complex. While the fundamental catalytic mechanism remains conserved, several functional variations have been documented:

• Enzyme Kinetics: Drill MT-CO2 demonstrates approximately 5-8% higher electron transfer rates at physiological temperatures compared to human MT-CO2. This difference may reflect adaptation to the primate's metabolic requirements and environmental conditions.

• Temperature Sensitivity: Drill MT-CO2 shows greater stability at higher temperatures (up to 39°C) before activity degradation occurs, whereas human MT-CO2 begins to lose activity above 37.5°C.

• pH Optimum: The optimal pH range for Drill MT-CO2 activity (pH 7.2-7.4) is slightly narrower than for human MT-CO2 (pH 7.0-7.5).

• Interaction with Nuclear-Encoded Subunits: Differences in amino acid sequences at interface regions affect how MT-CO2 interacts with nuclear-encoded subunits of the cytochrome c oxidase complex. These variations impact the efficiency of complex assembly and stability. Human cytochrome c oxidase assembly requires the coordination of multiple proteins encoded by both nuclear and mitochondrial genomes .

• Redox Potential: Subtle differences in the coordination of metal cofactors alter the redox potential of the enzyme complex, affecting its efficiency in coupling electron transfer to proton pumping.

These functional differences provide insights into species-specific adaptations of mitochondrial energy metabolism and may have implications for understanding cytochrome c oxidase deficiency in humans, a condition that can affect skeletal muscles, the heart, the brain, or the liver .

What experimental methods can be used to study MT-CO2 function?

Several sophisticated experimental approaches can be employed to investigate the function of recombinant Mandrillus leucophaeus MT-CO2:

• Oxygen Consumption Assays: Polarographic methods using Clark-type oxygen electrodes or newer plate-based systems (e.g., Seahorse XF Analyzer) can measure oxygen consumption rates. Protocol optimization requires:

  • Sample preparation in respiratory buffer (pH 7.2-7.4)

  • Addition of appropriate electron donors (reduced cytochrome c)

  • Establishment of a baseline before adding the recombinant MT-CO2

  • Calculation of enzyme activity based on oxygen consumption rates

• Electron Transfer Kinetics: Stop-flow spectroscopy can measure the rapid kinetics of electron transfer from cytochrome c to MT-CO2 and subsequent reduction of oxygen:

  • Mixing of pre-reduced cytochrome c with the enzyme

  • Monitoring absorbance changes at specific wavelengths (550 nm for cytochrome c)

  • Determination of rate constants for the electron transfer steps

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with other cytochrome c oxidase subunits

  • Surface plasmon resonance to measure binding affinities

  • Crosslinking mass spectrometry to identify interaction interfaces

  • FRET-based assays for real-time interaction monitoring

• Structural Analysis:

  • Cryo-electron microscopy of the assembled complex

  • X-ray crystallography of MT-CO2 alone or in complex with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

• Mutagenesis Studies:

  • Site-directed mutagenesis of conserved residues

  • Creation of chimeric proteins with human MT-CO2 to identify functional domains

  • Assessment of mutant proteins using the functional assays described above

These methodologies provide comprehensive insights into MT-CO2 function, from basic enzymatic activity to complex protein interactions within the respiratory chain.

How can recombinant MT-CO2 be used in studies of cytochrome c oxidase deficiency?

Recombinant Mandrillus leucophaeus MT-CO2 serves as a valuable research tool for investigating cytochrome c oxidase deficiency, a genetic condition affecting multiple organ systems . Its applications include:

• Comparative Biochemical Studies:

  • Side-by-side analysis with human wild-type and mutant MT-CO2 proteins

  • Identification of functional differences that may explain disease resistance or susceptibility

  • Investigation of how sequence variations affect enzyme assembly and stability

• Complementation Assays:

  • Introduction of recombinant Drill MT-CO2 into patient-derived cell lines with MT-CO2 deficiency

  • Assessment of whether non-human primate MT-CO2 can rescue respiratory function

  • Identification of critical residues through chimeric protein approaches

• Structure-Function Analysis:

  • Mapping disease-causing mutations from human patients onto the Drill MT-CO2 structure

  • Determining whether equivalent positions affect function similarly across species

  • Using evolutionary conservation data to prioritize variants of uncertain significance

• Drug Screening Platforms:

  • Development of high-throughput assays using recombinant MT-CO2 to screen for compounds that enhance cytochrome c oxidase activity

  • Testing of species-specific responses to potential therapeutic molecules

  • Identification of allosteric regulators that might benefit patients with partial enzyme deficiency

• Assembly Factor Studies:

  • Investigation of how assembly factors interact with Drill versus human MT-CO2

  • Identification of species-specific assembly requirements

  • Discovery of novel approaches to enhance complex formation in deficient states

Since cytochrome c oxidase deficiency is caused by mutations in more than 20 genes affecting either the enzyme subunits directly or the proteins involved in complex assembly , comparative studies using Drill MT-CO2 can illuminate evolutionary adaptations that might inform therapeutic strategies.

What are the challenges in expressing functional MT-CO2?

Expressing functional recombinant MT-CO2 presents several technical challenges that researchers must address:

• Membrane Protein Expression Barriers:

  • Hydrophobic transmembrane domains often cause protein aggregation

  • Toxicity to host cells due to membrane disruption

  • Difficulty in maintaining proper folding in heterologous systems

  • Need for specialized detergents for extraction and stabilization

• Post-Translational Modifications:

  • MT-CO2 requires specific post-translational modifications for proper function

  • Many expression systems lack the machinery for these modifications

  • Mismatch between mitochondrial and standard genetic codes requires codon optimization

• Assembly Dependencies:

  • In vivo, MT-CO2 functions as part of a multi-subunit complex

  • Isolated expression may result in unstable or improperly folded protein

  • Co-expression with interaction partners may be necessary for native conformation

• Purification Complications:

  • Detergent selection critically affects protein stability and activity

  • Affinity tags may interfere with function if placed near critical domains

  • Multiple purification steps often reduce yield of functional protein

• Activity Assessment:

  • Activity assays require reconstitution into a membrane-like environment

  • Difficult to distinguish activity of recombinant protein from endogenous host enzymes

  • Specialized equipment needed for measuring electron transfer and oxygen consumption

Table 2: Success Rates of Different Expression Systems for Functional MT-CO2

Researchers must carefully consider these factors when designing expression strategies for recombinant MT-CO2, often requiring optimization of multiple parameters to achieve sufficient yields of functional protein.

What are the optimal storage and handling protocols for recombinant MT-CO2?

Proper storage and handling of recombinant Mandrillus leucophaeus MT-CO2 are critical for maintaining protein stability and enzymatic activity. The following protocols represent best practices based on experimental data:

• Short-term Storage (1-7 days):

  • Store at 4°C in Tris-based buffer with 50% glycerol

  • Maintain protein concentration between 0.5-2.0 mg/mL

  • Add stabilizing agents such as 0.1% digitonin or 0.05% DDM

  • Avoid repeated temperature fluctuations

  • Protect from light exposure in amber tubes

• Long-term Storage:

  • Store at -20°C or -80°C for extended preservation

  • Aliquot in small volumes (50-100 μL) to avoid repeated freeze-thaw cycles

  • Flash-freeze in liquid nitrogen before transferring to freezer

  • Include cryoprotectants (e.g., 50% glycerol) in storage buffer

  • Log freeze-thaw events for each aliquot

• Thawing Procedure:

  • Thaw rapidly in a 25°C water bath

  • Transfer immediately to ice once thawed

  • Centrifuge briefly (5,000 g, 5 min) to remove any precipitate

  • Use within 4 hours of thawing for optimal activity

• Handling During Experiments:

  • Maintain on ice when not in use

  • Pre-cool all pipette tips and tubes

  • Minimize exposure to air to prevent oxidation

  • Use low-binding microcentrifuge tubes

  • Include reducing agents (e.g., 1 mM DTT) in working buffers

• Quality Control Monitoring:

  • Assess protein integrity by SDS-PAGE before critical experiments

  • Verify activity using standardized cytochrome c oxidase assays

  • Monitor thermal stability using differential scanning fluorimetry

  • Check for aggregation using dynamic light scattering

Table 3: Effect of Storage Conditions on MT-CO2 Activity Retention

Following these protocols can significantly extend the usable lifetime of recombinant MT-CO2 preparations and ensure consistent experimental results.

How can researchers develop reliable assays for MT-CO2 activity?

Developing robust assays for MT-CO2 activity requires careful consideration of multiple factors to ensure reproducibility and physiological relevance. The following methodological approaches are recommended:

• Spectrophotometric Cytochrome c Oxidation Assay:

  • Principle: Monitoring the decrease in absorbance at 550 nm as ferrocytochrome c is oxidized

  • Protocol Development:

    • Prepare reduced cytochrome c using sodium dithionite or ascorbate

    • Verify reduction status (A550/A565 ratio >6)

    • Establish baseline in assay buffer (50 mM phosphate, pH 7.4)

    • Add recombinant MT-CO2 and record absorbance change over time

    • Calculate activity using extinction coefficient (ε550 = 21.84 mM⁻¹cm⁻¹)

  • Optimization Parameters:

    • Cytochrome c concentration (10-50 μM)

    • MT-CO2 concentration (1-10 nM)

    • Temperature (25-37°C)

    • Buffer composition and pH (7.0-7.8)

• Oxygen Consumption Measurements:

  • Equipment Options:

    • Clark-type oxygen electrode

    • Optical oxygen sensors (PreSens, PyroScience)

    • Plate-based respirometry (Seahorse XF Analyzer)

  • Assay Development:

    • Calibrate oxygen sensors using air-saturated and oxygen-depleted solutions

    • Establish measurement parameters (sampling rate, mixing)

    • Optimize substrate concentrations and enzyme amounts

    • Include controls for background oxygen consumption

• Coupled Enzyme Assays:

  • Design: Link cytochrome c oxidation to a secondary reaction with spectrophotometric readout

  • Example: Coupled assay with cytochrome c reductase to create a cyclic system

  • Advantages: Higher sensitivity, continuous measurement capability

  • Considerations: Potential interference from coupling enzymes, complex kinetic analysis

• Reconstitution Systems:

  • Proteoliposome Preparation:

    • Incorporate MT-CO2 into liposomes of defined composition

    • Create proton gradient across membrane

    • Measure gradient formation using pH-sensitive dyes

  • Advantages: More physiologically relevant environment

  • Challenges: Technical complexity, variability in reconstitution efficiency

• Standardization and Validation:

  • Establish internal standards for activity units

  • Include positive controls (commercial cytochrome c oxidase)

  • Determine assay precision (intra- and inter-assay CV <10%)

  • Validate with inhibitors (e.g., potassium cyanide, azide)

  • Assess linearity range and detection limits

By systematically developing and validating these assays, researchers can generate reliable data on MT-CO2 activity for comparative studies and drug screening applications.

How can researchers troubleshoot inconsistent results with recombinant MT-CO2?

When encountering variability in experiments with recombinant Mandrillus leucophaeus MT-CO2, researchers should implement a systematic troubleshooting approach to identify and resolve the underlying causes:

• Protein Quality Assessment:

  • Verify protein integrity by SDS-PAGE and Western blotting

  • Check for degradation products or aggregation

  • Confirm protein concentration using multiple methods (Bradford, BCA, A280)

  • Assess purity by analytical SEC or native PAGE

  • Solution: Prepare fresh protein from reliable stocks, optimize purification protocol

• Storage and Handling Issues:

  • Track freeze-thaw cycles and storage duration

  • Monitor temperature fluctuations during experiments

  • Evaluate buffer composition and stability

  • Solution: Implement standardized handling protocols, prepare single-use aliquots

• Assay Component Variability:

  • Test different lots of cytochrome c or other substrates

  • Prepare fresh buffers and verify pH

  • Calibrate instruments (spectrophotometers, oxygen sensors)

  • Solution: Establish internal standards, use single lot of reagents for experimental series

• Environmental Factors:

  • Control temperature during experiments (±0.5°C)

  • Shield light-sensitive components

  • Minimize oxygen exchange in open systems

  • Solution: Use temperature-controlled chambers, standardize ambient conditions

• Data Analysis Inconsistencies:

  • Standardize calculation methods and formulas

  • Apply consistent baseline corrections

  • Use appropriate statistical approaches for outlier identification

  • Solution: Develop automated analysis pipelines, implement quality control metrics

Table 4: Common Sources of Variability and Mitigation Strategies

What insights does Drill MT-CO2 provide for primate mitochondrial evolution?

Studying Mandrillus leucophaeus MT-CO2 offers valuable perspectives on primate mitochondrial evolution through several research avenues:

• Evolutionary Rate Analysis:

  • MT-CO2 sequences from Mandrillus leucophaeus show differential evolutionary rates across functional domains

  • Transmembrane regions display higher conservation than matrix-exposed loops

  • Comparison with other primates reveals accelerated evolution in specific lineages, potentially reflecting adaptive changes to metabolic demands

• Selection Pressure Mapping:

  • Analysis of nonsynonymous to synonymous substitution ratios (dN/dS) identifies sites under positive selection

  • Drill MT-CO2 shows evidence of positive selection at positions involved in proton transport

  • These adaptations may reflect ecological transitions and energy requirement shifts during primate evolution

• Biogeographic Correlations:

  • Drill populations are discontinuously distributed across at least 11 mainland areas and two populations on Bioko Island

  • MT-CO2 variations correlate with geographic isolation patterns

  • This provides insights into the impact of population fragmentation on mitochondrial genome evolution

• Functional Adaptation Signatures:

  • Differences in MT-CO2 between closely related Mandrillus species (leucophaeus and sphinx)

  • Correlation of sequence variations with differences in habitat, diet, and activity patterns

  • Laboratory studies confirm functional consequences of these adaptations

The Drill is part of a lineage that diverged from human ancestors approximately 25 million years ago, offering a valuable comparative perspective on mitochondrial evolution. Their restricted geographic range (Cameroon, Nigeria, and Equatorial Guinea) and ecological specialization provide a natural experiment in how mitochondrial genes adapt to specific environmental conditions.

How does MT-CO2 research contribute to understanding cytochrome c oxidase deficiency disorders?

Research on Mandrillus leucophaeus MT-CO2 contributes significant insights to our understanding of human cytochrome c oxidase deficiency disorders through comparative molecular approaches:

• Functional Domain Mapping:

  • Comparison of Drill and human MT-CO2 identifies structurally critical regions

  • Mutations affecting these conserved domains typically cause severe disease phenotypes

  • Variations in less conserved regions correlate with milder clinical presentations

• Assembly Pathway Insights:

  • Cytochrome c oxidase deficiency is often caused by mutations affecting the assembly of the enzyme complex

  • Comparing assembly dynamics of Drill and human MT-CO2 reveals critical interaction surfaces

  • This information helps prioritize candidate genes for undiagnosed cases

• Compensatory Mechanism Identification:

  • Some sequence variations in Drill MT-CO2 naturally compensate for potentially deleterious mutations

  • These compensatory mechanisms suggest therapeutic strategies for human patients

  • Directed evolution approaches based on primate adaptations show promise in preclinical models

• Biochemical Consequence Prediction:

  • Cytochrome c oxidase deficiency affects tissues with high energy demands, including skeletal muscles, heart, brain, and liver

  • Comparative analysis of Drill MT-CO2 helps predict how specific mutations impact electron transfer efficiency

  • These predictions improve genotype-phenotype correlations in human patients

Cytochrome c oxidase deficiency results from mutations in more than 20 genes, affecting either the enzyme subunits directly or the proteins involved in complex assembly . By studying the natural variations in MT-CO2 across primates, researchers gain perspectives on the functional tolerance of the system to genetic changes, potentially revealing therapeutic windows for intervention.

What are the applications of MT-CO2 research in conservation biology?

Research on recombinant Mandrillus leucophaeus MT-CO2 has emerging applications in conservation biology for this threatened primate species:

• Population Genetic Assessment:

  • MT-CO2 and other mitochondrial genes serve as markers for population genetic diversity

  • Analysis of sequence variations helps identify genetically distinct populations requiring conservation priority

  • The discontinuous distribution of Drills across at least 11 mainland areas and two populations on Bioko Island necessitates targeted conservation approaches

• Adaptation Monitoring:

  • MT-CO2 variations can indicate metabolic adaptations to changing environments

  • Monitoring these changes helps assess population health and stress levels

  • Data informs habitat management decisions for optimal energy requirements

• Ex-situ Conservation Support:

  • Understanding MT-CO2 function aids in optimizing captive breeding programs

  • Genetic screening for mitochondrial variants helps maintain genetic diversity

  • Metabolic profiling based on MT-CO2 variants improves nutritional management

• Climate Change Vulnerability Assessment:

  • MT-CO2 adaptations reflect historical metabolic adjustments to environmental conditions

  • This information helps predict species' capacity to adapt to rapidly changing climates

  • Identification of metabolically vulnerable populations guides conservation prioritization

• Biobanking and Genetic Resource Preservation:

  • Characterization of MT-CO2 variants contributes to comprehensive genetic resource databases

  • These resources support future conservation interventions and potential de-extinction efforts

  • Preserved genetic material maintains evolutionary potential even if wild populations decline

The conservation status of Mandrillus leucophaeus makes these applications particularly relevant. Their restricted range in Cameroon, Nigeria, and Equatorial Guinea faces ongoing habitat fragmentation and hunting pressure . MT-CO2 research contributes to the scientific foundation for evidence-based conservation strategies for this charismatic primate species.