Recombinant Rat Cytochrome b-c1 complex subunit Rieske, mitochondrial (Uqcrfs1), partial

What is the function of the Rieske iron-sulfur protein in mitochondrial respiration?

The Rieske iron-sulfur protein (RISP or Uqcrfs1) serves as a catalytic core subunit of the ubiquinol-cytochrome c oxidoreductase (cytochrome b-c1 complex, Complex III) in the mitochondrial electron transport chain. This complex is essential for oxidative phosphorylation, cooperating with other respiratory complexes to transfer electrons derived from NADH and succinate to molecular oxygen. The Rieske protein contains a critical [2Fe-2S] iron-sulfur cluster that cycles between two conformational states during catalysis, facilitating electron transfer from ubiquinol bound in the Q(0) site in cytochrome b to cytochrome c1 .

Specifically, the cytochrome b-c1 complex catalyzes electron transfer from ubiquinol to cytochrome c while simultaneously translocating protons across the mitochondrial inner membrane. During this process, known as the Q cycle, 2 protons are consumed from the matrix, 4 protons are released into the intermembrane space, and 2 electrons are passed to cytochrome c . This proton translocation contributes to the electrochemical gradient that drives ATP synthesis.

How is the Rieske protein processed and incorporated into Complex III?

The incorporation of Uqcrfs1 represents the penultimate step in Complex III assembly. The protein undergoes proteolytic processing once incorporated into the Complex III dimer. One notable fragment, designated as subunit 9, corresponds to its mitochondrial targeting sequence (MTS) . This processing mechanism is essential for the correct insertion of Uqcrfs1 into the Complex III dimer.

Research has demonstrated that the persistence of Uqcrfs1-derived fragments can have detrimental effects on Complex III assembly and function. These fragments may prevent newly imported Uqcrfs1 from being properly processed and incorporated into the complex, negatively affecting its structural integrity and functional capacity .

What expression systems are most effective for producing recombinant Rieske protein?

E. coli represents the predominant expression system for recombinant Rieske protein production. When designing expression constructs, researchers have achieved optimal results by creating fusion proteins. For instance, studies have demonstrated successful overexpression of both full-length and truncated Rieske proteins from Spinacia oleracea (spinach) fused to MalE in E. coli systems .

These fusion protein approaches yield significant expression levels, with 55-70% of the overexpressed fusion proteins found in the cytoplasm in soluble form. Using affinity chromatography (specifically amylose resin), researchers can obtain approximately 15 mg of electrophoretically pure protein from 1 liter of E. coli culture . This purified material is suitable for various downstream applications, including structural studies and functional reconstitution experiments.

What methods can be used to verify the identity and purity of recombinant Rieske protein?

Multiple analytical approaches should be employed to confirm the identity and assess the purity of recombinant Rieske protein preparations:

SDS-PAGE Analysis: This represents the primary method for purity assessment. High-quality preparations typically demonstrate >90% purity by SDS-PAGE . The observed molecular weight for the rat Rieske protein is approximately 23 kDa, which differs slightly from the calculated mass of 30 kDa, likely due to proteolytic processing or the influence of the protein's tertiary structure on electrophoretic mobility .

Western Blotting: Immunological verification using specific antibodies against Uqcrfs1 provides confirmation of protein identity. Multiple commercial antibodies are available that recognize epitopes in the human, mouse, and rat Rieske proteins .

Mass Spectrometry: For definitive identification, peptide mass fingerprinting or tandem mass spectrometry can be performed on tryptic digests of the purified protein, comparing observed peptide masses with theoretical values derived from the known sequence of rat Uqcrfs1 .

Spectroscopic Analysis: The presence of the characteristic [2Fe-2S] cluster can be verified through UV-visible spectroscopy, which should show absorption features typical of iron-sulfur proteins, and through EPR spectroscopy, which can confirm the electronic properties of the reconstituted cluster .

How does the absence of Rieske protein affect the assembly and function of respiratory complexes?

Studies using Rieske iron-sulfur protein knockout (RISP KO) cells have provided significant insights into the interdependence of respiratory complex assembly and function. In the absence of RISP, most remaining CIII subunits can still assemble into a large precomplex, but this structure lacks enzymatic activity . This finding indicates that while RISP is not essential for the initial assembly of the CIII structural scaffold, it is absolutely required for catalytic function.

More significantly, RISP deficiency has profound effects beyond CIII. RISP KO cells demonstrate decreased levels of Complex I (CI), Complex IV (CIV), and respiratory supercomplexes . When RISP is reintroduced into these knockout cells, not only is CIII activity restored, but the levels of active CI, CIV, and supercomplexes also increase . This demonstrates the critical interdependence of respiratory complexes and suggests that RISP plays an important role in the stabilization of supercomplexes.

What methods can be used to reconstitute the iron-sulfur cluster in recombinant Rieske proteins?

Reconstitution of the [2Fe-2S] cluster in recombinant Rieske apoprotein represents a critical step for obtaining functionally active protein. Enzymatic reconstitution using NifS-like proteins has proven particularly effective for this purpose. The NifS-like protein IscS from the cyanobacterium Synechocystis PCC 6803 can mediate the incorporation of 2Fe-2S clusters into both apoferredoxin and cyanobacterial Rieske apoprotein in vitro .

The reconstitution procedure typically involves:

• Incubation of the purified recombinant Rieske apoprotein with the IscS enzyme

• Addition of iron (usually as ferrous ammonium sulfate) and sulfur sources (typically cysteine)

• Inclusion of reducing agents (such as DTT) to maintain appropriate redox conditions

• Addition of pyridoxal phosphate as a cofactor for IscS activity

Following reconstitution, the presence and integrity of the incorporated [2Fe-2S] cluster can be verified using EPR spectroscopy. Both full-length and truncated Rieske fusion proteins have been successfully reconstituted using this approach, demonstrating characteristic EPR spectra consistent with the presence of a functional [2Fe-2S] cluster .

How can researchers investigate the conformational dynamics of the Rieske protein during electron transfer?

The Rieske protein undergoes significant conformational changes during electron transfer, cycling between two states as it transfers electrons from quinol to cytochrome c1 . Investigating these dynamics requires specialized techniques:

Site-directed Spin Labeling and EPR Spectroscopy: By introducing cysteine residues at strategic positions and labeling them with nitroxide spin labels, researchers can monitor conformational changes during electron transfer using continuous wave or pulsed EPR techniques.

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can identify regions of the protein that exhibit differential solvent accessibility during the catalytic cycle, providing insights into conformational dynamics.

Molecular Dynamics Simulations: Computational approaches can model the conformational transitions of the Rieske protein based on available structural data, generating testable hypotheses about the mechanisms of conformational coupling to electron transfer.

Single-Molecule FRET: By labeling specific domains of the Rieske protein with fluorescent donor and acceptor pairs, researchers can monitor distance changes during catalysis, potentially capturing transient conformational states that are difficult to observe using ensemble methods.

What is the impact of hypoxia on Rieske protein function and respiratory complex assembly?

Hypoxic conditions (1% O₂) have been found to influence respiratory complex assembly and function, with implications for Rieske protein-dependent processes . Under hypoxic conditions, cells may exhibit alterations in the assembly and stability of respiratory supercomplexes, potentially as an adaptive response to maintain energy production with limited oxygen availability.

Research investigating the relationships between hypoxia, Rieske protein function, and respiratory complex assembly should employ the following methodological approaches:

• Controlled oxygen tension cultivation systems to maintain precise hypoxic conditions

• Blue native PAGE analysis to assess changes in supercomplex formation

• Activity assays for individual respiratory complexes to determine functional consequences

• Proteomic analysis to identify potential hypoxia-induced post-translational modifications of the Rieske protein

• Transcriptomic and protein expression analysis to evaluate potential compensatory mechanisms

How can researchers study the role of Rieske protein in reactive oxygen species (ROS) production?

Cells lacking the Rieske iron-sulfur protein demonstrate altered reactive oxygen species (ROS) profiles, suggesting an important role for this protein in regulating mitochondrial ROS production . Methodological approaches to investigate this relationship include:

ROS-Specific Fluorescent Probes: Utilization of probes such as MitoSOX Red (for mitochondrial superoxide) and 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, for general cellular ROS) can provide quantitative assessment of ROS production in cells with normal versus altered Rieske protein expression.

Site-Directed Mutagenesis: Introduction of specific mutations in the Rieske protein, particularly those affecting the iron-sulfur cluster coordination or protein conformational dynamics, can help identify structural elements critical for ROS regulation.

Antioxidant Enzyme Activity Assays: Measurement of superoxide dismutase, catalase, and glutathione peroxidase activities in cells with modified Rieske protein expression can reveal compensatory mechanisms activated in response to altered ROS production.

Mitochondrial Membrane Potential Analysis: As ROS production is often linked to changes in mitochondrial membrane potential, techniques such as JC-1 or TMRM staining can provide insights into the bioenergetic consequences of Rieske protein dysfunction.

Genetic Complementation Studies: Reintroduction of wild-type or mutant Rieske proteins into knockout cells, followed by assessment of ROS production, can establish causative relationships between specific protein features and ROS generation.

What techniques can be used to study the incorporation of Rieske protein into Complex III during mitochondrial biogenesis?

Investigating the assembly pathway of Rieske protein into Complex III requires specialized techniques to track protein import, processing, and complex formation:

Pulse-Chase Experiments: Metabolic labeling of newly synthesized proteins with radioactive amino acids, followed by immunoprecipitation at various time points, can track the processing and incorporation of Rieske protein into Complex III.

In Organello Import Assays: Using isolated mitochondria and radiolabeled precursor proteins synthesized in vitro, researchers can study the import kinetics and processing events of the Rieske protein under controlled conditions.

Blue Native PAGE and 2D-PAGE: These techniques allow visualization of assembly intermediates and can track the incorporation of Rieske protein into the complete Complex III structure.

Inducible Expression Systems: Controlled expression of tagged Rieske protein can facilitate temporal studies of its incorporation into Complex III during biogenesis.

Proximity Labeling Techniques: Methods such as BioID or APEX2 proximity labeling can identify transient interaction partners of the Rieske protein during its assembly pathway, potentially revealing novel assembly factors.