Recombinant Pan troglodytes Cytochrome b-c1 complex subunit Rieske, mitochondrial (UQCRFS1)

What is UQCRFS1 and what is its role in mitochondrial function?

UQCRFS1, also known as the Rieske iron-sulfur protein (RISP), is an essential subunit of the Ubiquinol Cytochrome c Reductase (Complex III) in the mitochondrial respiratory chain. It functions as a critical component in electron transfer during oxidative phosphorylation. The protein contains an iron-sulfur cluster that accepts electrons from ubiquinol and transfers them to cytochrome c1, facilitating the proton translocation necessary for ATP generation . In Pan troglodytes, as in other mammals, UQCRFS1 is nuclear-encoded despite functioning in the mitochondria, requiring proper import mechanisms for its integration into Complex III .

How does Pan troglodytes UQCRFS1 compare structurally to human UQCRFS1?

The Pan troglodytes UQCRFS1 protein shows high sequence homology to its human counterpart, reflecting the close evolutionary relationship between chimpanzees and humans. The chimpanzee protein (UniProt: Q69BK5) consists of 274 amino acids with a molecular weight of approximately 29.7 kDa, similar to the human protein . The amino acid sequence includes a transmembrane domain and a characteristic Rieske iron-sulfur domain. The high conservation between species suggests critical functional constraints on this protein throughout primate evolution . The Pan troglodytes UQCRFS1 contains the signature [2Fe-2S] cluster binding motif that is essential for its electron transport function.

What are the key biochemical properties of recombinant Pan troglodytes UQCRFS1?

The recombinant Pan troglodytes UQCRFS1 protein has several important biochemical characteristics:

The protein exhibits stability in tris-based buffer with glycerol as a cryoprotectant. Repeated freeze-thaw cycles should be avoided to maintain protein integrity and activity .

What are the optimal conditions for handling and storing recombinant Pan troglodytes UQCRFS1?

For optimal handling of recombinant Pan troglodytes UQCRFS1, researchers should adhere to strict storage protocols to maintain protein integrity. The protein should be stored in a Tris-based buffer containing 50% glycerol at -20°C for routine storage or at -80°C for extended periods . Working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw cycles which can compromise protein structure and activity. When thawing frozen stock, gentle thawing on ice is recommended rather than rapid warming .

For experimental use, the protein is typically diluted in PBS or other physiologically relevant buffers. Researchers should avoid introducing contaminants by using sterile technique and consider adding protease inhibitors if degradation is a concern. The addition of reducing agents like DTT or β-mercaptoethanol (0.1-1 mM) may help maintain the integrity of the iron-sulfur cluster during experiments .

How should researchers validate the activity of recombinant UQCRFS1 prior to experimental use?

Validation of recombinant UQCRFS1 activity is crucial before incorporating it into complex experimental designs. A multi-tiered approach is recommended:

• Spectroscopic Analysis: The iron-sulfur cluster in UQCRFS1 has characteristic absorption peaks at approximately 330 nm and 460 nm. Monitoring these spectral features can confirm the presence of properly folded protein with intact Fe-S centers .

• Electron Transfer Capacity Assessment: The functional activity can be measured using a ubiquinol-cytochrome c reductase assay, where the rate of cytochrome c reduction is monitored spectrophotometrically at 550 nm. Active UQCRFS1 will facilitate electron transfer from ubiquinol to cytochrome c .

• Protein Integrity Confirmation: SDS-PAGE analysis under reducing and non-reducing conditions can verify protein size and purity, while western blotting using anti-UQCRFS1 antibodies can confirm identity .

• Thermal Shift Assays: These can assess protein stability and proper folding by monitoring the protein's denaturation profile across a temperature gradient .

For quantitative comparisons across experiments, researchers should establish standard curves relating protein concentration to activity measures and maintain consistent assay conditions .

How can researchers effectively study the incorporation of recombinant UQCRFS1 into the cytochrome b-c1 complex?

Studying the incorporation of recombinant UQCRFS1 into the cytochrome b-c1 complex requires sophisticated biochemical and biophysical approaches:

• Blue Native PAGE (BN-PAGE): This non-denaturing electrophoretic technique allows visualization of intact protein complexes. Researchers can monitor the integration of labeled recombinant UQCRFS1 into existing Complex III structures by combining the recombinant protein with isolated mitochondria or submitochondrial particles .

• Immunoprecipitation Assays: Using antibodies against other Complex III components, researchers can assess whether recombinant UQCRFS1 co-precipitates, indicating successful integration into the complex .

• Fluorescence Resonance Energy Transfer (FRET): By labeling recombinant UQCRFS1 and known Complex III components with appropriate fluorophores, researchers can detect physical interactions through energy transfer events when the proteins come into close proximity .

• Enzymatic Activity Reconstitution: Perhaps the most definitive approach involves depleting native UQCRFS1 from Complex III preparations and measuring the restoration of electron transport activity upon addition of recombinant protein. This can be monitored through oxygen consumption rates or cytochrome c reduction kinetics .

When conducting these experiments, it's essential to control for non-specific binding and ensure that observed interactions represent physiologically relevant associations rather than experimental artifacts .

What experimental approaches can differentiate between functional and structural roles of UQCRFS1 in Complex III?

Differentiating between the functional and structural contributions of UQCRFS1 to Complex III requires strategic experimental designs:

• Site-Directed Mutagenesis: Creating recombinant UQCRFS1 variants with mutations in:

  • Iron-sulfur cluster binding residues (affecting electron transfer)

  • Transmembrane domain residues (affecting structural integration)

  • Interface residues that contact other Complex III subunits

• Domain Swap Experiments: Replacing domains of Pan troglodytes UQCRFS1 with corresponding regions from divergent species can reveal which portions are critical for function versus structural integrity .

• Inhibitor Studies: Using specific inhibitors like stigmatellin or myxothiazol that block electron transfer at different points can help isolate the functional contribution of UQCRFS1 .

• Time-Resolved Spectroscopy: Monitoring electron transfer kinetics through the [2Fe-2S] cluster can separate electron transfer functions from structural roles .

• Partial Complex Reconstitution: Assembling subcomplexes with and without UQCRFS1 to determine which aspects of Complex III function depend specifically on this subunit .

The results from these approaches should be integrated with structural data from cryo-electron microscopy or X-ray crystallography to build a comprehensive model of UQCRFS1's multifaceted roles in Complex III .

How can researchers effectively compare UQCRFS1 function across different primate species?

Comparing UQCRFS1 function across primates requires a systematic approach that accounts for both sequence and functional conservation:

• Sequence-Based Comparative Analysis:

  Species	UniProt ID	Sequence Identity to Human (%)	Key Divergent Residues
  Homo sapiens	P47985	100	Reference
  Pan troglodytes	Q69BK5	~99	Limited to a few positions
  Gorilla gorilla	Similar high identity	~98-99	Primarily in non-functional regions
  Pongo pygmaeus	High conservation	~95-97	Few differences in peripheral regions
  Other primates	Various	Decreasing with phylogenetic distance	More extensive differences

• Functional Heterologous Expression: Expressing UQCRFS1 from different primate species in a common cellular background (such as UQCRFS1-knockout cell lines) allows direct comparison of functional parameters including:

  • Oxygen consumption rates

  • ROS production

  • Complex III assembly efficiency

  • Electron transfer kinetics

• Cross-Species Complex III Reconstitution: Mixing subunits from different species to create hybrid complexes can reveal compatibility constraints and functional differences .

• Molecular Dynamics Simulations: Computational approaches can predict how subtle sequence differences might affect protein motion and interactions within Complex III .

These comparative approaches can reveal adaptive changes in UQCRFS1 that may correlate with metabolic differences across primate species, potentially reflecting environmental adaptations throughout evolutionary history .

What are the key considerations when using Pan troglodytes UQCRFS1 as a model for human mitochondrial disease research?

When using Pan troglodytes UQCRFS1 as a model for human mitochondrial disease research, researchers should consider several important factors:

• Sequence Conservation: While the high sequence similarity (~99%) between human and chimpanzee UQCRFS1 makes it an excellent model, researchers must identify and account for any amino acid differences, particularly in regions associated with human pathogenic mutations .

• Post-Translational Modification Differences: Subtle differences in phosphorylation, acetylation, or other modifications between species may affect protein function or regulation despite high sequence conservation .

• Nuclear-Mitochondrial Genetic Interactions: The interaction between nuclear-encoded UQCRFS1 and mitochondrial-encoded Complex III components (like cytochrome b) may differ between species due to co-evolutionary constraints .

• Metabolic Context Differences: Chimpanzees and humans have different metabolic profiles and energetic demands that may influence the phenotypic consequences of UQCRFS1 variants .

• Disease Modeling Limitations: Some human UQCRFS1-related disorders may involve species-specific interactions with other cellular components or metabolic pathways not conserved in chimpanzees .

To address these considerations, researchers should incorporate complementary approaches using both species' proteins and validate findings in appropriate cellular models that recapitulate the relevant metabolic context .

How can researchers utilize recombinant UQCRFS1 to investigate mitochondrial supercomplex formation?

Investigating mitochondrial supercomplex formation using recombinant UQCRFS1 requires sophisticated techniques that can capture dynamic protein-protein interactions:

• Fluorescently-Tagged UQCRFS1 for Live Imaging: Recombinant UQCRFS1 with genetically encoded fluorescent tags (ensuring the tag doesn't disrupt function) can be used in live-cell microscopy to monitor supercomplex assembly dynamics in real time .

• Crosslinking Mass Spectrometry (XL-MS): Chemical crosslinking of recombinant UQCRFS1 with other respiratory complex components followed by mass spectrometry analysis can map interaction interfaces within supercomplexes .

• Cryo-Electron Microscopy Reconstitution: Purified recombinant UQCRFS1 can be used in reconstitution experiments with other respiratory complex components to build supercomplexes for structural analysis by cryo-EM .

• Surface Plasmon Resonance (SPR): This technique can quantify binding kinetics and affinities between recombinant UQCRFS1 and components of Complex I or IV to understand supercomplex formation principles .

• Nanodiscs Technology: Incorporating recombinant UQCRFS1-containing Complex III into lipid nanodiscs with other respiratory complexes allows controlled analysis of supercomplex assembly in defined membrane environments .

Through these approaches, researchers can determine the specific contributions of UQCRFS1 to supercomplex stability, electron channeling efficiency, and how these processes might be affected by disease-associated mutations .

What methodological approaches are most effective for studying the iron-sulfur cluster assembly in UQCRFS1?

Studying iron-sulfur cluster assembly in UQCRFS1 requires specialized techniques that can track this complex biochemical process:

• Anaerobic Protein Expression and Purification: The oxygen-sensitive nature of iron-sulfur clusters necessitates anaerobic techniques during recombinant protein production to obtain UQCRFS1 with intact clusters or cluster-free protein for reconstitution studies .

• Mössbauer Spectroscopy: This technique can characterize the oxidation state and local environment of iron atoms in the [2Fe-2S] cluster of UQCRFS1, providing detailed information about cluster integrity and electronic structure .

• EPR Spectroscopy: Electron paramagnetic resonance can detect the paramagnetic states of the iron-sulfur cluster, offering insights into its redox properties and structural environment .

• In Vitro Fe-S Cluster Reconstitution: Using purified apo-UQCRFS1 (without clusters) and iron-sulfur cluster assembly components (like NFS1, ISCU, FDX1, FDX2) allows monitoring of cluster insertion kinetics and efficiency .

• Pulse-Chase Experiments with Isotope-Labeled Iron: These experiments can track the incorporation of labeled iron into newly synthesized UQCRFS1, revealing the dynamics of cluster assembly in cellular contexts .

• Interaction Studies with Fe-S Assembly Machinery: Techniques like co-immunoprecipitation or BioID proximity labeling can identify the specific components of the iron-sulfur cluster assembly machinery that interact with UQCRFS1 during cluster insertion .

These approaches collectively provide a comprehensive understanding of how the crucial iron-sulfur cofactor is assembled into UQCRFS1, which is essential for its electron transfer function in Complex III .