Recombinant Papio anubis Cytochrome c oxidase subunit 2 (MT-CO2)

What is Cytochrome c oxidase subunit 2 and what role does it play in cellular respiration?

Cytochrome c oxidase subunit 2 (MT-CO2) is a mitochondrial-encoded protein that forms part of the catalytic core of cytochrome c oxidase (COX), the terminal enzyme in the electron transport chain. In the olive baboon (Papio anubis) as in other mammals, MT-CO2 plays a crucial role in cellular respiration by facilitating electron transfer from cytochrome c to the catalytic center where oxygen is reduced to water. This enzyme represents the final stage of the respiratory chain where molecular oxygen is reduced to form water in a reaction coupled to energy conservation . MT-CO2 contains the Cu_A center, which serves as the primary electron acceptor from cytochrome c before electrons are transferred to heme a in subunit 1.

Why is Papio anubis an important model organism for MT-CO2 research?

Papio anubis (olive baboon) represents a valuable non-human primate model for research for several reasons:

• Phylogenetic proximity to humans makes baboons more relevant for translational research than rodent models

• Baboons and humans share significant physiological similarities in their immune and metabolic systems

• Unlike macaques, baboons express CD28 on nearly all CD4+ T lymphocytes in their effector memory cell compartment, similar to humans

• The olive baboon provides useful insights into age-related changes to the immune system and understanding mechanisms of immunosenescence

These characteristics make baboons particularly suitable for studying proteins like MT-CO2 in contexts that more closely approximate human biology.

How is MT-CO2 evolutionarily conserved across primate species?

While the search results don't specifically detail MT-CO2 evolution, studies of other cytochrome c oxidase subunits provide insights into the evolutionary patterns likely applicable to MT-CO2:

• Cytochrome c oxidase shows evidence of anthropoid-specific adaptive evolution, with selected amino acid changes occurring on specific lineages

• The three-subunit core of cytochrome c oxidase (including MT-CO2) is highly conserved across species, reflecting its essential role in cellular respiration

• Amino acid changes in one subunit can influence interactions with other subunits, creating co-evolutionary patterns

For instance, in studies of COX5A, researchers identified "four positively selected sites with posterior probabilities > 0.95, two showed changes in their physicochemical properties" . Similar evolutionary patterns may exist in MT-CO2, particularly at sites that interact with other subunits or with cytochrome c.

What is the structure of Papio anubis MT-CO2 and how does it differ from human MT-CO2?

Based on search result , the Papio anubis MT-CO2 protein has the following properties:

Table 2: Recommended Purification Approach

For recombinant MT-CO2, special consideration should be given to maintaining the integrity of the Cu_A center during purification by potentially including copper ions in buffers or performing reconstitution post-purification.

How can the functional activity of recombinant Papio anubis MT-CO2 be assessed?

Several methods can be employed to verify the functionality of recombinant MT-CO2:

• Spectroscopic analysis: UV-visible spectroscopy to assess characteristic absorbance features of the heme and copper centers

• Electron transfer assays: Using artificial electron donors and acceptors to measure electron transfer rates

• Binding assays: As mentioned in search result , "The biological activity was determined by its binding ability in a functional ELISA," which could be adapted for MT-CO2 to assess interaction with cytochrome c

• Reconstitution studies: Incorporation into liposomes or with other COX subunits to measure oxygen consumption activity

• Circular dichroism: To evaluate proper folding and secondary structure content

How do mutations in MT-CO2 affect electron transfer and proton pumping?

Based on cytochrome c oxidase research described in search results and :

• During the catalytic cycle, electrons from cytochrome c are accepted by the Cu_A center in MT-CO2 and then transferred to heme a and finally to the binuclear center (BNC) where oxygen reduction occurs

• Changes in the physicochemical properties of amino acid residues, particularly charged residues, can significantly impact electron transfer efficiency: "the charge neutralizations are parallel to those observed at the binding site for cytochrome c on COX in anthropoids, where the binding site changes reduced the electrostatic interaction between the docked molecules"

• Proton transfer during the catalytic cycle involves "one proton from matrix (chemical proton) are translocated into BNC, while one proton is pumped into IMS by conserved proton channels"

• Mutations affecting the structure around the Cu_A center or electron transfer pathways can impair the coupling between electron transfer and proton pumping, potentially leading to reduced energy conservation or increased ROS production

What regulatory considerations apply to research with recombinant Papio anubis proteins?

Research involving recombinant Papio anubis proteins, including MT-CO2, must comply with several regulatory requirements:

• NIH Guidelines: "Compliance with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules is mandatory for every institution that receives NIH funding for research involving recombinant DNA"

• Institutional Biosafety Committee (IBC) approval: Required for experiments involving recombinant DNA from mammalian sources

• Biosafety levels: Generally, work with purified recombinant proteins requires Biosafety Level 1, but expression systems may require Biosafety Level 2, especially when using viral vectors or mammalian cells

• Animal care regulations: When sourcing material from Papio anubis, additional regulations regarding animal care apply, as noted in search result : "All experiments in mice and non-human primates were performed in accordance with the recommendations of the Institutional Ethical Guidelines"

How does the Cu_A center in MT-CO2 coordinate copper atoms and what role do they play in enzyme function?

The Cu_A center in MT-CO2 is a binuclear copper center that serves as the primary electron acceptor from cytochrome c. Based on the structural and functional information in search results:

• The Cu_A center contains two copper atoms in a unique arrangement that facilitates rapid electron transfer

• Copper atoms are coordinated by histidine and cysteine residues, forming a structure that can accept and donate electrons while minimizing reorganization energy

• As described in result , this arrangement is part of the "binuclear center (BNC) involved in the reduction of oxygen to water"

• The copper atoms in the Cu_A center undergo redox changes (Cu+/Cu2+) during electron transfer, accepting electrons from cytochrome c and transferring them to heme a in subunit 1

• Proper incorporation of copper atoms during protein expression and purification is essential for functional activity of recombinant MT-CO2

What are common challenges in expressing and purifying functional MT-CO2?

Researchers often encounter several challenges when working with recombinant MT-CO2:

• Membrane protein solubility: MT-CO2 is a membrane protein, which can present solubility issues during expression and purification

• Metal center incorporation: Ensuring proper formation of the Cu_A center with correctly incorporated copper atoms

• Protein folding: Achieving native-like folding, especially in prokaryotic expression systems that lack the chaperones and folding machinery of eukaryotic cells

• Stability concerns: According to search result , repeated freeze-thaw cycles should be avoided, and working aliquots should be stored at 4°C for up to one week

• Reconstitution challenges: When attempting to incorporate purified MT-CO2 into functional assays or with other COX subunits

How can protein yield and stability be optimized when working with recombinant MT-CO2?

Based on information from related recombinant Papio anubis proteins:

Table 3: Optimization Strategies for Recombinant MT-CO2

What analytical methods are most appropriate for characterizing recombinant Papio anubis MT-CO2?

Several complementary analytical approaches can be used to thoroughly characterize recombinant MT-CO2:

• SDS-PAGE: For assessing purity and apparent molecular weight; "Purity: Greater than 90% as determined by SDS-PAGE"

• Western blot analysis: For specific detection and verification of the recombinant protein

• Mass spectrometry: For accurate molecular weight determination and identification of post-translational modifications

• UV-visible spectroscopy: To analyze the characteristic absorption spectra of the heme and copper centers

• Circular dichroism: For assessment of secondary structure content and proper folding

• Functional ELISA: As mentioned in search result , biological activity can be "determined by its binding ability in a functional ELISA"

• Electron transfer assays: To confirm the protein's ability to transfer electrons in reconstituted systems

How does Papio anubis MT-CO2 compare to MT-CO2 from other model organisms?

While the search results don't provide direct comparisons, we can infer from evolutionary patterns of cytochrome c oxidase:

• Primate MT-CO2 sequences show high conservation of functional domains due to strong selective pressure on this essential respiratory protein

• As noted in search result regarding another COX subunit: "changes in COX5Ap are rare among placental mammals outside the primate clade," suggesting similar conservation patterns may exist for MT-CO2

• Differences between species might be concentrated at interfaces with other subunits or with cytochrome c, potentially affecting the efficiency of electron transfer

• The baboon model provides advantages over other non-human primate models in certain contexts: "in baboons (Papio anubis), in contrast with macaques, CD28-CD4+ T lymphocytes are barely detectable in peripheral blood and that, in baboons, like in man, TEM cells are CD28+"

What in vitro systems are available for studying MT-CO2 function in the context of the complete COX complex?

Several experimental systems can be employed:

• Reconstituted proteoliposomes: Purified recombinant subunits can be assembled into proteoliposomes to measure electron transfer and proton pumping activities

• Cell-free expression systems: As used for other membrane proteins, allowing co-expression of multiple subunits

• Heterologous expression in yeast: Yeast mutants lacking endogenous COX components can be complemented with Papio anubis genes

• HUVEC co-culture systems: While primarily used for immunological studies as described in search result , similar co-culture approaches might be adapted for studying respiratory chain components in more physiologically relevant contexts

How might MT-CO2 studies in Papio anubis contribute to understanding human mitochondrial disorders?

Research on Papio anubis MT-CO2 has significant translational potential:

• As a non-human primate model, Papio anubis provides closer evolutionary proximity to humans than rodent models, making findings more directly applicable to human mitochondrial biology

• Studies of MT-CO2 variants could provide insights into human mitochondrial disorders caused by mutations in MT-CO2 or other COX subunits

• The baboon model could be used to study age-related changes in mitochondrial function, as baboons are also being used to study "age-related changes to the immune system and understanding mechanisms of immunosenescence"

• Techniques developed for recombinant expression and characterization of Papio anubis MT-CO2 could be applied to study human MT-CO2 variants identified in patients with mitochondrial disorders