Recombinant Gorilla gorilla gorilla Cytochrome c oxidase subunit 2 (MT-CO2)

What is the evolutionary significance of MT-CO2 in gorillas and other higher primates?

Cytochrome c oxidase subunit 2 has undergone remarkable evolutionary changes in the anthropoid lineage. Research demonstrates that monkeys and apes, including gorillas, have experienced a nearly two-fold increase in the rate of amino acid replacements in MT-CO2 compared to other primates . This accelerated evolution appears concentrated at the amino terminal end of the protein, while functionally critical amino acids remain generally conserved across primate species .

The evolutionary pattern suggests selective pressure on respiratory chain components in the lineage leading to great apes, potentially related to metabolic adaptations supporting increased brain size . This hypothesis is strengthened by evidence that cytochrome c oxidase plays a significant role in controlling respiratory chain activity and global energy production . When investigating this phenomenon, researchers should employ comparative sequence analysis across diverse primate species to establish evolutionary rates and identify specific amino acid replacements unique to the gorilla lineage.

How do amino acid replacements in gorilla MT-CO2 affect its interaction with cytochrome c?

The interaction between MT-CO2 and cytochrome c in higher primates, including Gorilla gorilla gorilla, shows distinct biochemical properties compared to non-primate mammals. Particularly significant are the replacements of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 in higher primates . These substitutions provide a molecular explanation for the poor enzyme kinetics observed in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals .

For researchers investigating this interaction, it is recommended to employ recombinant protein expression systems that maintain native folding and post-translational modifications of both proteins. Experimental approaches should include:

Comparing wild-type gorilla MT-CO2 with site-directed mutants where key residues are restored to the ancestral state allows quantification of their specific contribution to altered interaction properties.

What expression systems are most suitable for producing functional recombinant gorilla MT-CO2?

Although the search results don't provide specific information about gorilla MT-CO2 expression, we can infer from search result that E. coli expression systems represent one potential approach, as used for other gorilla recombinant proteins. When working with mitochondrial membrane proteins like MT-CO2, researchers should consider:

• Codon optimization for the expression host's translational machinery

• Addition of solubility-enhancing tags (such as MBP or SUMO)

• Expression at reduced temperatures (16-20°C) to improve protein folding

• Optimization of membrane-mimetic environments for protein extraction and purification

For more native-like protein production, mammalian expression systems may offer advantages for proper folding and post-translational modifications. Regardless of the system chosen, verification of proper folding and assembly should be confirmed through spectroscopic methods assessing heme incorporation and functional assays measuring electron transfer activity.

How can researchers effectively measure the functional activity of recombinant MT-CO2 in vitro?

Functional assessment of recombinant Gorilla MT-CO2 requires incorporation into the complete cytochrome c oxidase complex (Complex IV). Based on methodological approaches used in cytochrome c oxidase research, investigators should employ:

• Polarographic oxygen consumption assays using Clark-type electrodes to measure the rate of oxygen reduction

• Spectrophotometric assays monitoring the oxidation of reduced cytochrome c at 550 nm

• Measurement of proton pumping efficiency using pH-sensitive dyes or electrodes

• Analysis of electron transfer rates through artificial electron donors and acceptors

When conducting these experiments, researchers should control for:

Accurate interpretation requires calculating enzyme kinetic parameters under various conditions to assess substrate affinity and catalytic efficiency, with particular attention to differences from human MT-CO2 that may reflect evolutionary adaptations.

What are the implications of the accelerated evolution of MT-CO2 for primate brain metabolism?

The accelerated evolution of MT-CO2 in higher primates has been proposed to be linked to the energy metabolism rearrangement needed for brain enlargement . This hypothesis is supported by the fact that cytochrome c oxidase has a large control coefficient on oxidative phosphorylation activity and its activity is tightly regulated by several cellular mechanisms .

For researchers investigating the connection between MT-CO2 evolution and brain metabolism, methodological approaches should include:

• Comparative analyses of brain-specific expression patterns of MT-CO2 across primates

• Oxygen consumption measurements in neuronal tissues expressing different MT-CO2 variants

• Assessment of ATP production efficiency under varying oxygen conditions

• Analysis of whether gorilla-specific MT-CO2 variants alter the control coefficient of COX on oxidative phosphorylation

Research should address whether the substitutions at positions 114 and 115 in higher primate MT-CO2 affect the enzyme's response to changing oxygen levels, potentially reflecting adaptations to different metabolic requirements in brain tissue compared to other organs .

How can researchers distinguish between neutral evolution and adaptive selection in gorilla MT-CO2?

Distinguishing between neutral evolution and adaptive selection in Gorilla MT-CO2 requires rigorous statistical and comparative genomic approaches. Based on evolutionary analysis methodologies used for respiratory chain components , researchers should implement:

• Codon-based maximum likelihood methods to calculate dN/dS ratios and test for positive selection

• Branch-site tests to identify specific codons under selection in the gorilla lineage

• Population genetic analyses using data from multiple gorilla individuals to identify signals of selective sweeps

• Comparative analysis across multiple primate species, as performed in studies examining 25 different primates

The table below summarizes key considerations for evolutionary analysis of MT-CO2:

Integration of these computational approaches with functional characterization provides a robust framework for distinguishing adaptive changes from neutral evolution in gorilla MT-CO2.

What is the relationship between MT-CO2 evolution and regulatory mechanisms of cytochrome c oxidase?

The evolution of MT-CO2 should be considered in the context of the complex regulatory mechanisms controlling cytochrome c oxidase activity. Research indicates that COX is targeted by cell signaling pathways through phosphorylation of specific residues, which can decisively regulate enzyme activity . Additionally, cytochrome c oxidase is unique among respiratory chain complexes in having tissue-specific and developmentally-regulated isoforms .

To investigate how MT-CO2 evolution relates to these regulatory mechanisms in Gorilla gorilla gorilla, researchers should:

• Identify potential phosphorylation sites in gorilla MT-CO2 that may differ from human MT-CO2

• Examine whether tissue-specific expression patterns of MT-CO2 vary between gorillas and other primates

• Investigate how gorilla-specific amino acid substitutions might affect the interaction with regulatory proteins

• Assess whether the accelerated evolution of MT-CO2 correlates with changes in regulatory subunits of COX

This research would reveal whether evolutionary changes in gorilla MT-CO2 have influenced not only protein function but also its regulation in response to varying metabolic demands across tissues and developmental stages.

How do oxygen sensitivity mechanisms differ between gorilla MT-CO2 and other species?

While search result focuses primarily on COX4 isoforms rather than MT-CO2, it provides insights into how oxygen sensitivity varies across species. Unlike the oxygen-responsive elements found in mammals, the COX4-2 gene appeared unresponsive to low oxygen in non-mammalian models . This suggests species-specific adaptations in oxygen sensing mechanisms.

For researchers investigating oxygen sensitivity in relation to gorilla MT-CO2, methodological approaches should include:

• Analysis of oxygen binding kinetics using rapid-mixing stopped-flow spectroscopy

• Measurement of enzyme activity across a range of oxygen tensions (0.1-100% O₂)

• Comparison of oxygen affinity (Km for O₂) between human and gorilla MT-CO2

• Investigation of whether gorilla-specific amino acid substitutions affect oxygen binding channels or proton exit pathways

These experiments should be conducted under various physiological conditions to determine whether gorilla MT-CO2 exhibits unique adaptations in oxygen sensing that may reflect the species' ecological niche and metabolic requirements.

What structural techniques are most informative for studying recombinant gorilla MT-CO2?

To elucidate the structure of recombinant Gorilla MT-CO2 and its integration into the complete cytochrome c oxidase complex, researchers should employ a multi-technique approach:

• X-ray crystallography of the complete cytochrome c oxidase complex containing MT-CO2, aiming for resolution below 2.5 Å

• Cryo-electron microscopy (cryo-EM) as an alternative approach for structural determination without the need for crystallization

• Hydrogen-deuterium exchange mass spectrometry to identify regions with differential solvent accessibility

• Molecular dynamics simulations to predict functional implications of gorilla-specific amino acid differences

For comparative structural analysis, researchers should focus on:

These approaches can reveal how specific amino acid replacements in gorilla MT-CO2 alter protein structure and potentially impact function in comparison to other primates.

How should researchers approach comparative studies of MT-CO2 across different primate species?

When conducting comparative studies of MT-CO2 across primates, researchers should implement a systematic approach incorporating evolutionary, structural, and functional analyses. Based on methodologies used in previous studies examining 25 primate species , researchers should:

• Sample broadly across primate phylogeny, including representatives from hominoids, Old World monkeys, New World monkeys, tarsiers, and strepsirrhines

• Perform phylogenetic analysis to establish evolutionary relationships and identify lineage-specific changes

• Map amino acid substitutions onto structural models to predict functional implications

• Conduct in vitro functional studies comparing recombinant MT-CO2 from different species

Particular attention should be paid to:

• The nearly two-fold increase in amino acid replacement rates observed in monkeys and apes relative to other primates

• Conservation patterns of functionally important residues across species

• Correlation between MT-CO2 sequence changes and primate brain evolution

• Species-specific adaptations that may reflect different ecological niches and metabolic requirements

This comprehensive approach allows researchers to contextualize gorilla MT-CO2 characteristics within the broader framework of primate evolution.

What methods are most effective for studying the assembly of MT-CO2 into the complete cytochrome c oxidase complex?

Studying the assembly of Gorilla MT-CO2 into the complete cytochrome c oxidase complex requires specialized techniques addressing both the timing and efficiency of this process. Based on approaches used in cytochrome c oxidase research, investigators should employ:

• Blue native polyacrylamide gel electrophoresis to visualize assembly intermediates

• Pulse-chase experiments with radiolabeled amino acids to track the kinetics of incorporation

• Proximity labeling approaches to identify transient interaction partners during assembly

• Immunoprecipitation of assembly factors followed by mass spectrometry

Comparative studies between human and gorilla MT-CO2 should examine:

This research would reveal whether evolutionary changes in gorilla MT-CO2 have influenced not only protein function but also the complex biogenesis process that is essential for respiratory chain activity.

How can researchers investigate the coevolution of cytochrome c and MT-CO2 in gorillas?

The coevolution of cytochrome c and MT-CO2 represents a fascinating example of molecular adaptation. Studies suggest that as little as seven million years is sufficient to change key amino acids that can disrupt harmonious assembly and function of these interacting proteins . To investigate this coevolution in gorillas, researchers should:

• Perform comparative sequence analysis of both cytochrome c and MT-CO2 across primates

• Identify correlated substitutions that may represent compensatory changes

• Use statistical coupling analysis to detect coevolving residue networks

• Conduct cross-species functional assays combining cytochrome c and MT-CO2 from different primates

The experimental approach should include:

This research would illuminate whether the accelerated evolution observed in both proteins represents a case of coevolution driven by the need to maintain optimal functional interactions despite rapid sequence changes.