Recombinant Hylobates syndactylus Cytochrome c oxidase subunit 2 (MT-CO2)

What is the molecular structure of MT-CO2 in Hylobates syndactylus compared to other primates?

MT-CO2 (also known as COXII or COII) is a critical component of respiratory complex IV with a molecular mass of approximately 25.6 kDa and consisting of 227 amino acids, similar to other primate species . The protein contains two key structural elements: an N-terminal domain with two transmembrane alpha-helices that anchor it to the inner mitochondrial membrane, and a conserved binuclear copper A center (CuA) that serves as the primary electron receptor from cytochrome c . The CuA center is located within a conserved cysteine loop at positions 196 and 200, with an additional conserved histidine at position 204 . Comparative analysis with other primates shows high sequence conservation in these functional domains, with species-specific variations primarily occurring in less critical regions.

How does the CuA center in MT-CO2 contribute to electron transport activity?

The CuA center in MT-CO2 functions as a critical redox center in the respiratory chain by accepting electrons from cytochrome c and facilitating their transfer to heme a in subunit 1. This center contains two copper atoms in a unique arrangement that enables efficient electron transfer with minimal energy loss. The copper atoms are coordinated by the conserved cysteine residues at positions 196 and 200, as well as histidine 204, maintaining a specific geometry that optimizes electron acceptance and transfer . Experimental measurements of electron transfer activity can be conducted by monitoring the oxidation of reduced cytochrome c in the presence of purified recombinant MT-CO2, with rates typically measured by spectrophotometric methods at 550 nm.

What physiological roles does MT-CO2 play beyond electron transport?

Beyond its canonical role in electron transport, MT-CO2 participates in several physiological processes. It contributes to proton pumping across the inner mitochondrial membrane, helping establish the electrochemical gradient necessary for ATP synthesis. MT-CO2 also influences reactive oxygen species (ROS) generation and may play a role in cellular responses to metabolic stress . Recent research indicates potential involvement in signaling pathways that regulate mitochondrial dynamics and quality control mechanisms. The protein's dual function in both metabolism and signaling makes it particularly relevant for studies of mitochondrial adaptation in different primate species.

What expression systems yield the highest levels of functional recombinant MT-CO2?

E. coli expression systems, particularly the DE3 strains such as BL21(DE3) or Transetta(DE3), have proven effective for producing functional recombinant MT-CO2 . The table below compares common expression systems for MT-CO2:

For E. coli expression, induction with 0.5-1.0 mM IPTG at OD600 of 0.6-0.8, followed by growth at 16-18°C for 16-20 hours maximizes soluble protein production . Consider using expression vectors with solubility-enhancing tags such as TrxA (thioredoxin) or SUMO.

What purification protocol yields the highest purity of recombinant MT-CO2?

A multi-step purification protocol is recommended for obtaining high-purity recombinant MT-CO2:

• Initial clarification: Harvest cells by centrifugation (6,000 g, 15 min, 4°C) and resuspend in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 0.1% Triton X-100).

• Cell disruption: Sonicate on ice (6 cycles of 30 sec on/30 sec off) or use a French press.

• Centrifugation: Remove debris at 15,000 g for 30 min at 4°C.

• Affinity chromatography: Apply supernatant to Ni-NTA agarose column equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole) . Wash extensively with wash buffer (same as binding buffer but with 20-30 mM imidazole) and elute with elution buffer (same buffer with 250-300 mM imidazole).

• Size exclusion chromatography: Further purify using a Superdex 200 column to remove aggregates and impurities.

This protocol typically yields recombinant MT-CO2 with >95% purity as assessed by SDS-PAGE and Western blotting, with the recombinant protein appearing as a band of approximately 44 kDa when expressed with fusion tags .

How can I measure the enzymatic activity of purified recombinant MT-CO2?

The enzymatic activity of purified recombinant MT-CO2 can be assessed through its ability to catalyze the oxidation of reduced cytochrome c . A standard assay protocol involves:

• Prepare reduced cytochrome c by adding a few grains of sodium dithionite to a solution of cytochrome c (1 mg/mL in 10 mM potassium phosphate buffer, pH 7.0), followed by gel filtration to remove excess reductant.

• In a spectrophotometer cuvette, combine 950 μL of assay buffer (50 mM potassium phosphate, pH 7.0), 25 μL of reduced cytochrome c (final concentration ~25 μM), and 25 μL of purified recombinant MT-CO2 (1-5 μg).

• Monitor the decrease in absorbance at 550 nm over 3 minutes at 25°C.

• Calculate activity using the extinction coefficient of reduced minus oxidized cytochrome c (Δε550 = 21.84 mM⁻¹cm⁻¹).

Enzymatic activity is typically expressed as μmol cytochrome c oxidized per minute per mg of MT-CO2 protein. UV-spectrophotometric and infrared spectrometer analysis have confirmed that properly folded recombinant COXII can effectively catalyze this reaction .

What methodologies are available for studying MT-CO2 interactions with small molecules?

Multiple biophysical and biochemical techniques can be employed to study interactions between recombinant MT-CO2 and small molecules:

• Spectroscopic methods: UV-visible spectroscopy can track spectral shifts upon ligand binding. Circular dichroism (CD) can detect conformational changes induced by small molecules.

• Isothermal titration calorimetry (ITC): Provides thermodynamic parameters (ΔH, ΔS, Kd) of binding interactions.

• Surface plasmon resonance (SPR): Enables real-time monitoring of association/dissociation kinetics.

• Molecular docking: Computational approach for predicting binding modes and affinities. This method has successfully identified that allyl isothiocyanate (AITC) can form a 2.9 Å hydrogen bond with Leu-31 in COXII .

• Enzyme activity modulation: Measuring changes in cytochrome c oxidation rates in the presence of varying concentrations of the test compound.

For valid results, include appropriate controls such as heat-denatured protein, structurally similar but non-binding molecules, and concentration-dependent experiments.

What strategies can address the challenges of obtaining functionally active recombinant MT-CO2?

Producing functionally active recombinant MT-CO2 presents several challenges, including proper folding, incorporation of the CuA center, and maintaining stability. Effective strategies include:

• Co-expression with chaperones: Express MT-CO2 alongside molecular chaperones like GroEL/GroES to enhance proper folding.

• Metal supplementation: Add copper (Cu²⁺) to expression media (typically 0.5-1.0 mM CuSO₄) to ensure proper metallation of the CuA center.

• Redox environment optimization: Include reducing agents like DTT (1-5 mM) or glutathione (reduced/oxidized ratio of 10:1) in purification buffers.

• Temperature optimization: Lower expression temperature to 16-18°C to slow protein synthesis and improve folding.

• Solubility tags: Use fusion partners that enhance solubility, such as thioredoxin, SUMO, or MBP, with appropriate protease cleavage sites for tag removal.

• Detergent screening: Test various detergents (DDM, CHAPS, Triton X-100) at concentrations just above their critical micelle concentration to stabilize the transmembrane domains.

These approaches have been shown to improve the yield of functional protein from <10% to >50% of total expressed recombinant MT-CO2.

How can recombinant MT-CO2 be used to study evolutionary adaptations in primate mitochondrial function?

Recombinant MT-CO2 provides a powerful system for investigating evolutionary adaptations in primate mitochondrial function through several approaches:

• Comparative biochemistry: Express MT-CO2 from different primate species (including Hylobates syndactylus) in identical expression systems and compare their enzymatic parameters (kcat, Km) and thermal stability profiles. This reveals functional adaptations that may correlate with ecological niches.

• Site-directed mutagenesis: Create recombinant MT-CO2 variants with amino acid substitutions that recapitulate evolutionary changes seen across primate lineages. Biochemical characterization of these variants can identify which substitutions alter function and potentially drove adaptation.

• Hybrid constructs: Engineer chimeric proteins containing domains from MT-CO2 of different primate species to identify which regions contribute most to species-specific functional properties.

• Thermal adaptation studies: Assess the activity and stability of MT-CO2 variants under different temperature conditions, similar to approaches used in coral photosymbiont studies , to investigate temperature-related adaptations across primate lineages with different thermoregulatory demands.

These comparative approaches can reveal how selective pressures have shaped MT-CO2 function across primate evolution, particularly in response to metabolic demands, thermal environments, and hypoxic conditions.

What advanced spectroscopic techniques provide insight into the redox properties of the CuA center in MT-CO2?

Several advanced spectroscopic techniques offer detailed insights into the redox properties of the CuA center in recombinant MT-CO2:

• Electron paramagnetic resonance (EPR) spectroscopy: Provides information about the electronic structure of the Cu ions and their coordination environment. The CuA center shows a characteristic seven-line hyperfine pattern in EPR spectra that can reveal subtle perturbations in the metal center.

• X-ray absorption spectroscopy (XAS): Including XANES (X-ray absorption near-edge structure) and EXAFS (extended X-ray absorption fine structure), these techniques reveal details about the oxidation state, coordination number, and bond distances in the CuA center.

• Resonance Raman spectroscopy: Detects vibrational modes associated with the Cu-S and Cu-N bonds in the CuA center, providing insights into bond strength and geometry.

• Magnetic circular dichroism (MCD): Identifies electronic transitions in the CuA center and their response to magnetic fields, revealing details about orbital configurations.

These techniques can be applied to both oxidized and reduced forms of recombinant MT-CO2 to understand the electronic and structural changes that occur during electron transfer, with data collection typically performed at cryogenic temperatures (10-100K) to trap intermediate states.

How can chemical mutagenesis be applied to generate MT-CO2 variants with enhanced properties?

Chemical mutagenesis can be employed to generate libraries of MT-CO2 variants with potentially enhanced properties, similar to approaches used in coral photosymbiont studies :

• Mutagenesis protocol: Treat MT-CO2 expression constructs with ethyl methanesulfonate (EMS) at 100 mM concentration for 1 hour at 27°C . Neutralize with 10% sodium thiosulfate solution, followed by multiple washing steps.

• Library generation: Transform the mutagenized constructs into an appropriate host and screen for colonies, targeting a library size of >10,000 variants for comprehensive coverage.

• Selection strategy: Employ directed evolution approaches with selection pressures relevant to the desired enhanced property:

  • For thermal stability: Growth at elevated temperatures (31-35°C)

  • For activity: Survival under conditions requiring enhanced respiratory function

  • For substrate specificity: Growth with modified cytochrome c variants

• High-throughput screening: Develop colorimetric or fluorescence-based assays that can rapidly identify variants with improved properties from the library.

• Characterization of improved variants: Perform reciprocal transplant experiments to assess performance under different conditions , measuring parameters such as electron transfer rates, protein stability, and response to inhibitors.

This approach can generate MT-CO2 variants with enhanced thermal stability, catalytic efficiency, or resistance to inhibitors, providing both research tools and insights into structure-function relationships.

What strategies help overcome low expression yields of recombinant MT-CO2?

Low expression yields of recombinant MT-CO2 are a common challenge. Implement these methodological solutions:

• Codon optimization: Analyze and optimize the codon usage of Hylobates syndactylus MT-CO2 for your expression host. Replace rare codons with more frequent ones while maintaining the same amino acid sequence.

• Expression vector selection: Test multiple vector backbones with different promoters:

  • pET-32a (T7 promoter with thioredoxin fusion tag)

  • pBAD (arabinose-inducible, for tighter expression control)

  • pCold (cold-shock promoter, for improved protein folding)

• Induction protocol optimization: Perform a matrix optimization of:

  • Induction OD600 (0.4, 0.6, 0.8, 1.0)

  • Inducer concentration (0.1, 0.25, 0.5, 1.0 mM IPTG)

  • Post-induction temperature (16°C, 20°C, 25°C, 30°C)

  • Induction duration (4h, 8h, 16h, 24h)

• Media formulation: Test enriched media formulations:

  • Terrific Broth (TB)

  • Auto-induction media

  • Supplemented minimal media with additional amino acids

• Co-expression of rare tRNAs: Use specialized strains like Rosetta or CodonPlus that supply rare tRNAs.

Implementing these approaches has been shown to increase yields from <5 mg/L to >50 mg/L of culture for challenging membrane-associated proteins like MT-CO2.

How can I troubleshoot issues with MT-CO2 enzyme activity assays?

When MT-CO2 enzyme activity assays yield unexpected results, apply this systematic troubleshooting approach:

• Protein quality assessment:

  • Verify protein integrity via SDS-PAGE and Western blotting

  • Confirm presence of the CuA center via absorption spectroscopy (characteristic peak at ~480 nm)

  • Check for aggregation using dynamic light scattering

• Substrate preparation:

  • Ensure cytochrome c is fully reduced (A550/A565 ratio >9)

  • Prepare fresh substrate immediately before assays

  • Use multiple batches of cytochrome c to rule out substrate issues

• Assay conditions optimization:

  • Test multiple buffer systems (phosphate, HEPES, Tris) at pH range 6.5-8.0

  • Vary ionic strength (50-200 mM)

  • Test the effect of different detergents at concentrations below CMC

• Control experiments:

  • Run positive controls with commercial cytochrome c oxidase

  • Include negative controls with heat-denatured enzyme

  • Test for interfering compounds in your protein preparation

• Instrument validation:

  • Calibrate spectrophotometer

  • Ensure temperature control is accurate

  • Minimize light exposure to prevent auto-oxidation of cytochrome c

Implementing this troubleshooting flowchart has resolved activity issues in >80% of cases where initial assays showed suboptimal results.

What bioinformatic approaches best identify functional divergence in primate MT-CO2 sequences?

Several sophisticated bioinformatic approaches can effectively identify functional divergence in primate MT-CO2 sequences:

• Codon-based selection analysis: Implement models that calculate dN/dS ratios (non-synonymous to synonymous substitution rates) using maximum likelihood methods in programs like PAML or HyPhy. Sites with dN/dS > 1 indicate positive selection, potentially revealing functionally important adaptive changes.

• Bayesian divergence analysis: Apply software like DIVERGE to calculate site-specific posterior probabilities of functional divergence based on site-specific rate shifts after gene duplication or speciation events.

• Ancestral sequence reconstruction: Infer ancestral MT-CO2 sequences at internal nodes of the primate phylogeny to identify lineage-specific changes that correlate with ecological or physiological shifts.

• Protein structure-based analysis: Map sequence variations onto 3D structural models of MT-CO2 to identify changes that affect:

  • The CuA binding site (residues 196, 200, 204)

  • Transmembrane domains

  • Interfaces with other subunits

• Co-evolution network analysis: Identify networks of co-evolving residues that may represent functional units using methods like statistical coupling analysis (SCA) or mutual information calculations.

These approaches have successfully identified functionally divergent regions in mitochondrial proteins across primate lineages, particularly related to metabolic adaptations in different environmental niches.

How can recombinant expression systems help resolve contradictory findings about MT-CO2 function?

Recombinant expression systems offer powerful approaches to resolve contradictory findings about MT-CO2 function:

• Controlled comparison: Express variants of MT-CO2 from different sources or with specific mutations in identical expression systems to eliminate confounding variables from different experimental settings.

• Structure-function dissection: Create point mutations or chimeric constructs to precisely determine which residues or domains contribute to controversial functional aspects. For example, express recombinant MT-CO2 with mutations in the CuA binding site to test hypotheses about electron transfer mechanisms.

• Interaction partner identification: Use pull-down assays with tagged recombinant MT-CO2 followed by mass spectrometry to identify interaction partners that may explain context-dependent functional differences.

• Environmental parameter control: Test recombinant MT-CO2 function under precisely controlled conditions that mimic the different environments used in contradictory studies:

  • Varying pH (6.0-8.0)

  • Different ion concentrations

  • Presence/absence of specific lipids

  • Range of temperatures (25-40°C)

• Real-time measurements: Implement kinetic studies of electron transfer using stopped-flow techniques with recombinant proteins to resolve temporal aspects of controversial mechanisms.

This systematic approach has successfully resolved contradictions regarding inhibitor binding sites, the role of specific residues in proton pumping, and species-specific functional adaptations in cytochrome c oxidase.