Recombinant Scaphirhynchus platorynchus Cytochrome c oxidase subunit 2 (mt-co2)

What is Cytochrome c oxidase subunit 2 (mt-co2) and what is its role in cellular respiration?

Cytochrome c oxidase subunit 2 (mt-co2) is a critical component of Complex IV in the mitochondrial electron transport chain. As part of the cytochrome c oxidase (CcO) complex, it plays a vital role in cellular respiration by catalyzing the final step of electron transfer to oxygen. The COX2 subunit contains the binuclear CuA site, which is essential for receiving electrons from cytochrome c before transferring them to the catalytic core . This function is crucial for energy production in aerobic organisms, including the Scaphirhynchus platorynchus.

In the complete respiratory complex, mt-co2 works in concert with other subunits to pump protons across the inner mitochondrial membrane, contributing to the proton gradient that drives ATP synthesis. The metal centers within the protein, particularly the CuA site, are essential for this electron transfer function and require proper coordination during biogenesis to ensure efficient respiratory function .

Why is the Scaphirhynchus platorynchus (Shovelnose sturgeon) mt-co2 of interest to researchers?

The Shovelnose sturgeon represents an evolutionary interesting species for several reasons. These fish are found in the main-stem Mississippi and Missouri rivers and are generally associated with flowing water habitats . Studying the mt-co2 from this species can provide insights into:

• Evolutionary adaptations in energy metabolism related to specific environmental conditions

• Molecular mechanisms underlying the physiological responses to temperature variation, as juvenile sturgeons show significant differences in swimming performance at different temperatures

• Comparative biochemistry across fish species, particularly within ancient lineages like sturgeons

• Structure-function relationships in respiratory chain components across vertebrate evolution

The recombinant version of this protein allows researchers to study its structure, function, and properties in controlled laboratory conditions without the need for harvesting large numbers of these protected fish species.

What are the key structural features of the Scaphirhynchus platorynchus mt-co2 protein?

According to available data, the recombinant Scaphirhynchus platorynchus mt-co2 protein has the following key structural features:

• Full-length protein spanning amino acids 1-64

• N-terminal His tag for purification and detection purposes

• Amino acid sequence: MAHPSQLGFQDAASPVMEELXHFHDHTLMIVFLISTLVXYIIVAMVSTKLTNKYVLDSQE IEIV

• The presence of 'X' in the sequence indicates positions where the exact amino acid is uncertain

• Based on general cytochrome c oxidase structure, the protein would contain the binuclear CuA site essential for electron transfer

• The protein is expected to have transmembrane domains that anchor it within the inner mitochondrial membrane in its native state

The relatively short length (64 amino acids) compared to mammalian mt-co2 proteins suggests this may represent a functional domain rather than the complete protein, though the product is listed as "full length" in the available information .

How does mt-co2 from Scaphirhynchus platorynchus compare to that of other species?

While direct comparative analyses are not provided in the available data, several comparative aspects can be considered:

• Evolutionary context: As a sturgeon, Scaphirhynchus platorynchus represents one of the most ancient lineages of ray-finned fishes, potentially preserving ancestral features in respiratory proteins

• Environmental adaptation: The specific amino acid sequence may reflect adaptations to the flowing water habitats and temperature regimes experienced by this species

• Size considerations: At 64 amino acids, the described "full-length" protein appears shorter than typical mt-co2 proteins in other vertebrates, which may indicate functional or structural differences unique to this species

• Conserved functional domains: Despite potential differences, the CuA binding site would be expected to be highly conserved due to its essential role in electron transfer

What challenges are associated with expressing and purifying recombinant Scaphirhynchus platorynchus mt-co2?

Expressing recombinant Scaphirhynchus platorynchus mt-co2 involves several technical challenges:

• Expression system compatibility: The available recombinant protein is expressed in E. coli , which lacks the specialized machinery for post-translational modifications found in eukaryotic cells. This may affect protein folding, particularly for a protein normally expressed in mitochondria.

• Membrane protein solubility: As a component of the inner mitochondrial membrane in its native state, mt-co2 contains hydrophobic domains that can cause aggregation during expression and purification.

• Metal center assembly: The CuA site in mt-co2 requires proper copper incorporation, which may not occur efficiently in heterologous expression systems. In native systems, specialized metallochaperones coordinate this process .

• Structural integrity: Maintaining the proper three-dimensional structure during purification requires careful optimization of buffer conditions, detergents, and stabilizing agents.

• Storage and handling: The final lyophilized product requires specific reconstitution protocols and storage conditions to maintain activity .

How can researchers overcome protein misfolding issues when working with recombinant mt-co2?

To address protein misfolding challenges with recombinant mt-co2, researchers can implement several strategies:

• Optimization of expression conditions:

  • Lower induction temperatures (16-20°C) to slow protein synthesis and allow time for proper folding

  • Reduced inducer concentrations to prevent overwhelming the cellular folding machinery

  • Selection of specialized E. coli strains designed for membrane protein expression

• Co-expression approaches:

  • Co-express with chaperone proteins that assist with folding

  • Include metallochaperones that facilitate copper incorporation, similar to those mentioned in research on human cytochrome c oxidase

• Solubilization strategies:

  • Use mild detergents for membrane protein extraction

  • Incorporate lipid components that mimic the native membrane environment

• Proper reconstitution:

  • Follow the recommended protocol: reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol for stability during storage

  • Consider reconstitution into nanodiscs or liposomes for functional studies

• Quality assessment:

  • Use multiple techniques to verify proper folding (circular dichroism, fluorescence spectroscopy)

  • Confirm metal incorporation using spectroscopic methods

What are the implications of studying mt-co2 for understanding evolutionary adaptations in sturgeon species?

Studying mt-co2 from Scaphirhynchus platorynchus provides unique opportunities for understanding evolutionary adaptations:

• Environmental adaptation mechanisms: Shovelnose sturgeon inhabit flowing water environments and show distinct swimming behaviors . The structure and function of mt-co2 may reflect adaptations in energy metabolism supporting these behaviors.

• Temperature response: The critical swimming speed of juvenile shovelnose sturgeon decreases significantly from 36.9 cm/s at 20°C to 19.4 cm/s at 10°C . This temperature sensitivity may be reflected in structural adaptations in respiratory proteins like mt-co2.

• Comparative genomics approach: Analyzing the sequence and structure of mt-co2 across sturgeon species could reveal:

  • Conserved regions essential for basic respiratory function

  • Variable regions representing species-specific adaptations

  • Evolutionary rates of change in different protein domains

• Ancient lineage insights: As one of the oldest extant ray-finned fish lineages, sturgeons may preserve ancestral features in respiratory proteins that have been modified in more recently diverged teleost lineages.

How can protein-protein interaction studies with mt-co2 provide insights into respiratory chain complexes in fish?

Protein-protein interaction studies with Scaphirhynchus platorynchus mt-co2 can reveal:

• Assembly process: Studies in human systems show that cytochrome c oxidase biogenesis involves coordinated assembly of multiple subunits and incorporation of metal centers . Similar studies in sturgeon could reveal:

  • Fish-specific assembly factors

  • Temperature-dependent assembly mechanisms

  • Species-specific interactions

• Respiratory supercomplex formation: Cytochrome c oxidase often forms supercomplexes with other respiratory chain components.

  • Investigating these interactions in sturgeon could reveal adaptations in energy metabolism

  • Different supercomplex arrangements may reflect metabolic adaptations to the sturgeon's environment

• Regulatory interactions: By identifying proteins that interact with mt-co2 under different conditions (e.g., temperature, oxygen levels), researchers can understand:

  • How respiratory function is regulated in response to environmental variables

  • Species-specific control mechanisms for energy production

• Evolutionary conservation: Comparing interaction networks across fish species can reveal:

  • Conserved core interactions essential for respiratory function

  • Lineage-specific interactions that may represent adaptive changes

What are the optimal conditions for reconstituting lyophilized Scaphirhynchus platorynchus mt-co2?

Based on available product information, the optimal reconstitution protocol for lyophilized mt-co2 is:

• Initial preparation:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Allow the product to reach room temperature before opening

• Reconstitution steps:

  • Add deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL

  • Gently mix by inversion or very mild vortexing

  • Allow the protein to fully dissolve (15-30 minutes at room temperature)

• Long-term storage preparation:

  • Add glycerol to a final concentration of 5-50% (recommended default is 50%)

  • Aliquot into small volumes to avoid repeated freeze-thaw cycles

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

• Working solution handling:

  • Working aliquots can be stored at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles which can compromise protein integrity

These conditions help maintain the structural integrity and functional properties of the recombinant protein for experimental applications.

What analytical techniques are most effective for studying the structure and function of recombinant mt-co2?

Several complementary techniques can be employed to comprehensively characterize recombinant mt-co2:

For functional characterization, additional techniques include:

• Oxygen consumption assays when incorporated into liposomes

• Electron transfer kinetics measurements

• Redox potential determination

How can researchers validate the functionality of recombinant mt-co2 in in vitro experiments?

Validating the functionality of recombinant mt-co2 requires multiple approaches:

• Spectroscopic validation:

  • Confirm proper copper incorporation using UV-visible spectroscopy

  • Verify characteristic absorbance features of the CuA center

  • Use EPR to confirm the electronic structure of the metal center

• Electron transfer capability:

  • Measure electron transfer rates from reduced cytochrome c

  • Compare kinetic parameters with native enzyme when possible

  • Assess the effect of temperature on electron transfer rates to understand thermal adaptation

• Reconstitution experiments:

  • Incorporate into liposomes or nanodiscs

  • Combine with other cytochrome c oxidase subunits to recreate partial or complete complex

  • Measure oxygen consumption in reconstituted systems

• Structural integrity verification:

  • Use limited proteolysis to confirm proper folding

  • Apply biophysical techniques (CD, fluorescence) to assess structural stability

  • Compare structural parameters across temperature ranges relevant to the species' habitat

• Comparative analysis:

  • Compare functional parameters with those of mt-co2 from related species

  • Assess temperature dependence of activity in context of the species' thermal biology

What are the best approaches for studying mt-co2 in the context of complete respiratory chain complexes?

To study mt-co2 within the context of complete respiratory complexes:

• Complex isolation and characterization:

  • Use blue native polyacrylamide gel electrophoresis (BN-PAGE) to separate intact complexes

  • Apply mild detergent solubilization conditions to preserve native interactions

  • Use activity staining to identify functional complexes

• Interaction mapping:

  • Perform co-immunoprecipitation using anti-His antibodies to capture the tagged mt-co2

  • Identify interacting partners through mass spectrometry

  • Use crosslinking approaches to capture transient interactions

• Functional reconstitution:

  • Combine purified components to recreate partial or complete complexes

  • Incorporate into liposomes to measure vectorial proton pumping

  • Assess the effect of lipid composition on complex assembly and function

• Structural approaches:

  • Apply cryo-electron microscopy to visualize complete complex architecture

  • Use hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Create fluorescently labeled components to study assembly dynamics

• Comparative analysis:

  • Compare complex composition and activity across temperature ranges relevant to the species

  • Assess species-specific differences in complex stability and activity

How does the swimming performance of Scaphirhynchus platorynchus relate to the function of mt-co2?

The swimming performance of Scaphirhynchus platorynchus provides context for understanding the functional requirements of its respiratory proteins:

These behavioral observations suggest:

• Temperature adaptation: The significant decrease in swimming performance at lower temperatures indicates that respiratory chain components, including mt-co2, must function across a range of thermal conditions. This may be reflected in structural adaptations that maintain function at lower temperatures.

• Energy efficiency: The limited free swimming behavior at higher speeds suggests energy conservation mechanisms that may involve specialized regulation of respiratory chain activity, potentially including adaptations in mt-co2 structure or function.

• Metabolic specialization: The sturgeon's ability to maintain station in flowing water habitats may be supported by specialized energy production pathways that could involve unique features in respiratory chain components.

What unique adaptations might be reflected in the structure of mt-co2 in Scaphirhynchus platorynchus?

The structure of mt-co2 in Scaphirhynchus platorynchus likely reflects several adaptations:

• Temperature compensation: Given the significant effect of temperature on swimming performance , sturgeon mt-co2 may contain adaptations that:

  • Maintain electron transfer efficiency across temperature ranges

  • Alter copper binding properties to function at lower temperatures

  • Include structural flexibility that accommodates thermal variation

• Energy optimization: The amino acid sequence (MAHPSQLGFQDAASPVMEELXHFHDHTLMIVFLISTLVXYIIVAMVSTKLTNKYVLDSQE IEIV) may contain:

  • Modifications that enhance electron transfer efficiency

  • Structural elements that optimize interaction with other respiratory chain components

  • Regions that participate in regulatory interactions specific to sturgeon metabolism

• Evolutionary heritage: As an ancient fish lineage, sturgeon mt-co2 may preserve ancestral features that have been modified in more recently diverged teleosts, providing insight into the evolution of respiratory proteins in vertebrates.

• Habitat-specific adaptations: The flowing water habitat preference may be reflected in mt-co2 adaptations that support:

  • Sustained energy production for station-holding behavior

  • Rapid modulation of respiratory activity in response to current changes

  • Efficient oxygen utilization in various flow conditions

How does temperature affect the function of mt-co2 in Scaphirhynchus platorynchus compared to other fish species?

Temperature significantly affects Scaphirhynchus platorynchus physiology, as evidenced by the reduction in critical swimming speed from 36.9 cm/s at 20°C to 19.4 cm/s at 10°C . This temperature sensitivity likely extends to mt-co2 function:

• Comparative thermal sensitivity:

  • The ~47% reduction in swimming performance between 20°C and 10°C suggests substantial thermal sensitivity

  • This may indicate that sturgeon mt-co2 has evolved to function optimally at moderate temperatures

  • Compared to cold-adapted fish species, sturgeon mt-co2 may show different structural adaptations

• Molecular mechanisms of thermal adaptation:

  • Temperature effects on mt-co2 could involve changes in:

    • Protein flexibility and conformational dynamics

    • Metal center redox properties

    • Interaction strength with electron donors and acceptors

  • These adaptations would directly impact energy production capacity at different temperatures

• Experimental approaches to assess temperature effects:

  • Measuring electron transfer rates across temperature ranges

  • Comparing thermal stability of the protein structure

  • Assessing temperature effects on protein-protein interactions

  • Monitoring copper binding affinity at different temperatures

• Ecological context:

  • Understanding how mt-co2 function changes with temperature provides insight into:

    • Seasonal activity patterns of sturgeons

    • Habitat selection based on thermal preferences

    • Potential vulnerability to climate change impacts

What role does the His-tag play in the recombinant Scaphirhynchus platorynchus mt-co2 protein, and how might it affect function?

The recombinant Scaphirhynchus platorynchus mt-co2 contains an N-terminal His-tag that serves several purposes but may also impact function:

• Technical benefits:

  • Enables purification using metal affinity chromatography

  • Facilitates detection using anti-His antibodies

  • May enhance solubility of the recombinant protein

  • Provides a uniform handle for binding in interaction studies

• Potential functional impacts:

  • May interfere with N-terminal structural elements

  • Could affect folding kinetics or final conformation

  • Histidine residues might compete for copper binding with native binding sites

  • May alter interaction with other respiratory chain components

• Methodological considerations:

  • Control experiments comparing tagged and untagged versions

  • Use of protease cleavage sites to remove the tag after purification

  • Comparison of N-terminal versus C-terminal tag placement

  • Verification that metal binding properties are preserved

• Experimental design implications:

  • When studying metal binding, consider potential interference

  • For structural studies, tag removal may be necessary

  • In interaction studies, confirm that observed interactions are not tag-mediated

  • For functional reconstitution, assess whether the tag affects assembly

How does the amino acid sequence of Scaphirhynchus platorynchus mt-co2 contribute to its metal-binding properties?

The amino acid sequence of Scaphirhynchus platorynchus mt-co2 (MAHPSQLGFQDAASPVMEELXHFHDHTLMIVFLISTLVXYIIVAMVSTKLTNKYVLDSQE IEIV) contains several features relevant to metal binding:

• Potential metal-coordinating residues:

  • Histidine residues (H) are common copper ligands in proteins

  • Methionine (M) can also coordinate copper ions

  • Acidic residues (D, E) may participate in metal coordination networks

• CuA binding domain features:

  • In typical mt-co2 proteins, the CuA site involves a His-X-X-X-Cys motif

  • The sequence contains histidine residues that could participate in copper binding

  • The presence of 'X' in the sequence makes it difficult to confirm all potential binding residues

• Structural context:

  • Secondary structure elements (likely α-helices spanning the membrane) position coordinating residues

  • The three-dimensional arrangement of these residues creates the appropriate geometry for metal binding

  • Hydrophobic residues (L, I, V, F) likely form the membrane-spanning domains

• Species-specific adaptations:

  • Comparing this sequence with other species could reveal:

    • Conserved metal-binding residues

    • Sturgeon-specific substitutions that might alter metal affinity or redox properties

    • Adaptations that affect stability or function at different temperatures

What insights can be gained from comparing the 3D structure of mt-co2 across different fish species?

Comparative structural analysis of mt-co2 across fish species would provide valuable insights:

• Evolutionary conservation and divergence:

  • Identify core structural elements conserved across all fish lineages

  • Pinpoint regions that have diverged in specific lineages

  • Map the evolutionary trajectory of structural changes

• Structure-function relationships:

  • Correlate structural features with known physiological differences

  • Identify adaptations in metal-binding regions that might affect electron transfer efficiency

  • Understand how protein dynamics may differ across species adapted to different environments

• Environmental adaptation mechanisms:

  • Compare structures from species living in different thermal regimes

  • Analyze differences between species with different activity levels or metabolic rates

  • Identify structural features that correlate with habitat preferences

• Molecular basis of physiological differences:

  • Relate structural differences to observed variations in swimming performance

  • Understand how protein structure influences temperature sensitivity

  • Identify interactions with other proteins that might be species-specific

• Application to conservation and environmental monitoring:

  • Develop models predicting how respiratory proteins might respond to changing environmental conditions

  • Understand species vulnerability to environmental stressors at the molecular level

  • Guide conservation efforts by identifying critical molecular adaptations