Recombinant Ateles sp. Cytochrome c (CYCS)

What expression systems are most effective for recombinant Cytochrome c production?

Escherichia coli remains the most widely used expression system for recombinant cytochrome c proteins. When using appropriate expression vectors and growth conditions, E. coli can yield approximately 8-15 mg of purified cytochrome c per liter of culture. This yield is comparable across various species; for instance, recombinant Rhizopus arrhizus cytochrome c yields approximately 9 mg/L, similar to human cytochrome c (>8 mg/L), while horse cytochrome c can yield up to 15 mg/L . For optimal expression in E. coli, codon optimization may be necessary when expressing Ateles sp. cytochrome c due to potential codon usage differences between primates and bacteria.

What purification strategy provides the highest purity for recombinant Cytochrome c?

A three-step purification process has proven highly effective for obtaining pure cytochrome c: ammonium sulfate precipitation, followed by cation exchange chromatography, and finally gel filtration chromatography. This method has been successfully applied to both native and recombinant cytochrome c proteins. The purified protein typically elutes from the gel filtration column with an absorption ratio (A410/A280) of >4.0, indicating excellent purity . SDS-PAGE analysis typically reveals a single band at approximately 14 kDa, confirming the homogeneity of the preparation.

How can I confirm the identity and proper folding of purified recombinant Cytochrome c?

Multiple complementary methods should be employed to verify the identity and proper folding of recombinant cytochrome c:

• UV-Visible spectroscopy: Properly folded cytochrome c in the oxidized state shows characteristic absorption peaks at approximately 409 nm (Soret band) and 529 nm, while the reduced form exhibits prominent α and β peaks at 549 nm and 520 nm, respectively .

• Mass spectrometry: MALDI-MS can confirm the molecular weight and identity of the purified protein.

• Functional assays: Assess electron transfer capability, peroxidase activity, and caspase activation potential as functional verification.

• Redox potential measurement: The redox potential of properly folded cytochrome c typically falls within the range of 263-270 mV .

How does the redox potential of Cytochrome c correlate with its electron transport function?

The redox potential of cytochrome c is a critical determinant of its electron transport efficiency. Typically, mammalian cytochrome c proteins exhibit redox potentials around 260-270 mV. For instance, human and horse cytochrome c show redox potentials of 263.43 mV and 268.64 mV, respectively . This property is crucial for maintaining the appropriate electron flow direction in the mitochondrial electron transport chain.

Post-translational modifications, particularly phosphorylation, can significantly alter the redox potential. For example, phosphorylation of Tyr48 results in a decrease of 45 mV in rodent cytochrome c and 80 mV in human cytochrome c compared to their wild-type counterparts . Such modifications can regulate electron transport rates and may serve as physiological control mechanisms.

What methods are recommended for evaluating the apoptotic function of recombinant Cytochrome c?

The apoptotic function of recombinant cytochrome c can be evaluated using cell-free caspase activation assays. In this approach, purified cytochrome c is added to cell extracts (commonly HeLa cell extracts) containing Apaf-1 and procaspase-3. The activation of caspase-3 is then measured using specific substrates that release detectable products upon cleavage .

When comparing cytochrome c proteins from different species, it's important to note that their apoptotic potential can vary significantly. These differences often stem from variations in surface charge distribution, particularly the arrangement of positively charged lysine residues (positions 7, 8, 25, 39, and 72) that are critical for cytochrome c/Apaf-1 interaction . Site-directed mutagenesis studies have demonstrated that disruption of these residues can significantly reduce or completely abolish the ability of cytochrome c to activate caspases.

How can peroxidase activity of Cytochrome c be accurately measured and what factors influence this activity?

While cytochrome c is not primarily a peroxidase, it does exhibit peroxidase-like activity that can be measured and used as an indicator of its functional integrity. This activity is typically lower than that of dedicated peroxidases but is still a useful functional parameter to assess.

The peroxidase activity can be influenced by:

• Protein purity: Recombinant cytochrome c often shows higher Kcat and Vmax values compared to native preparations, likely due to higher purity .

• Structural integrity: The methionine coordination to the heme is crucial for proper function. The presence of the charge transfer band in UV-visible spectra indicates intact methionine coordination .

• Post-translational modifications: Phosphorylation and other modifications can alter the peroxidase activity of cytochrome c.

How do phosphorylation events regulate Cytochrome c function in cellular metabolism and apoptosis?

Cytochrome c undergoes several phosphorylation events that significantly impact its functions. Five key phosphorylation sites have been mapped and functionally characterized: Tyr97, Tyr48, Thr28, Ser47, and Thr58 . These modifications have several important consequences:

• Respiratory control: All five phosphorylations partially inhibit respiration, which appears to help maintain optimal intermediate mitochondrial membrane potentials and low ROS production under normal physiological conditions .

• Apoptotic regulation: Four of these phosphorylations inhibit the apoptotic functions of cytochrome c, suggesting a cytoprotective role for phosphorylated cytochrome c .

• Stress response: These phosphorylations are typically lost during stress conditions such as ischemia, resulting in maximal electron transport chain flux during reperfusion, which can lead to membrane potential hyperpolarization, excessive ROS generation, and ultimately apoptosis .

This pattern suggests that post-translational modifications of cytochrome c serve as a regulatory mechanism to optimize cellular energy production while minimizing oxidative damage under normal conditions.

What structural and functional changes occur in Cytochrome c following Tyr97 phosphorylation?

Phosphorylation of Tyr97 induces significant structural and functional alterations in cytochrome c:

• Spectral shifts: This modification shifts the characteristic 695 nm heme-iron-Met80 absorption band to 687 nm, indicating alterations in the heme environment .

• Kinetic changes: In reactions with cytochrome c oxidase, Tyr97-phosphorylated cytochrome c displays sigmoidal kinetics (rather than the hyperbolic kinetics seen with unphosphorylated cytochrome c), with higher Km values (5.5 μM compared to 2.5 μM for unphosphorylated cytochrome c) .

• Apoptotic function: Tyr97 phosphorylation inhibits the apoptotic function of cytochrome c, contributing to cell survival under normal physiological conditions .

These changes collectively suggest that Tyr97 phosphorylation represents an important regulatory mechanism that modulates both the electron transport and apoptotic functions of cytochrome c.

What are the key structural and functional differences between primate Cytochrome c and other mammalian species?

Primate cytochrome c proteins, including those from Ateles species, share high sequence homology with other mammalian cytochrome c proteins, but exhibit some notable differences:

• Tyrosine phosphorylation: Tyrosine phosphorylation is predominantly found in higher organisms and plays important roles in mitochondrial and cancer signaling . The effects of these phosphorylation events can vary between species; for example, Tyr48 phosphorylation reduces the redox potential by 80 mV in human cytochrome c but only by 45 mV in rodent cytochrome c .

• Surface charge distribution: Differences in the distribution of positively charged residues on the protein surface can affect interactions with binding partners, including cytochrome c oxidase and Apaf-1 .

• Apoptotic potential: The ability to activate caspases can vary between species due to differences in key interaction residues involved in Apaf-1 binding .

These differences reflect evolutionary adaptations that may fine-tune cytochrome c function to match the specific metabolic and regulatory requirements of different species.

How do redox properties compare between Ateles sp. Cytochrome c and other well-characterized cytochrome c proteins?

While specific data for Ateles sp. cytochrome c is limited in the provided search results, comparisons between other mammalian cytochrome c proteins provide insight into potential variations:

What experimental approaches can effectively evaluate the role of Cytochrome c in mitochondrial membrane potential regulation?

To investigate cytochrome c's role in regulating mitochondrial membrane potential, researchers can employ several complementary approaches:

• Phosphorylation studies: Analyze how different phosphorylation states of cytochrome c affect membrane potential using fluorescent probes like TMRM or JC-1 .

• Redox potential measurements: Correlate changes in cytochrome c redox potential (via site-directed mutagenesis or post-translational modifications) with changes in membrane potential.

• Electron transfer rate analysis: Measure the rate of electron transfer from cytochrome c to cytochrome c oxidase under various conditions, as this appears to be the rate-limiting step of the electron transport chain that regulates membrane potential .

• ROS production assessment: Monitor how modifications to cytochrome c affect ROS generation, which is closely linked to membrane potential.

These approaches can help elucidate how cytochrome c serves as a regulatory point in maintaining optimal mitochondrial function while preventing excessive membrane potential that could lead to ROS production.

How can researchers distinguish between the electron transport and apoptotic functions of Cytochrome c in experimental settings?

Differentiating between these dual functions requires specific experimental designs:

• Site-directed mutagenesis: Create mutants that selectively impair one function while preserving the other. For example, mutations in lysine residues 7, 8, 25, 39, and 72 can disrupt apoptotic function without necessarily affecting electron transport .

• Phosphomimetic studies: Generate phosphomimetic variants (e.g., Tyr48Glu, Tyr97Glu) that simulate phosphorylation states known to differentially affect electron transport and apoptotic functions .

• Compartmentalized assays: Develop assays that isolate mitochondrial electron transport from cytosolic apoptotic activation, allowing separate measurement of each function.

• Temporal analysis: In cellular systems, monitor the kinetics of cytochrome c release from mitochondria and correlate this with changes in electron transport efficiency and initiation of apoptosis.

These approaches can provide valuable insights into how these distinct functions are regulated independently or interdependently in various physiological and pathological contexts.