Recombinant Nicotiana tabacum Cytochrome b-c1 complex subunit Rieske-2, mitochondrial

What is Nicotiana tabacum Cytochrome b-c1 complex subunit Rieske-2, mitochondrial?

Cytochrome b-c1 complex subunit Rieske-2, mitochondrial (UniProt: P51132) is an iron-sulfur protein component of the respiratory chain complex III (ubiquinol-cytochrome c reductase) in tobacco (Nicotiana tabacum) mitochondria. This protein contains a unique [2Fe-2S] cluster and plays a critical role in electron transfer during oxidative phosphorylation. The protein is characterized by its distinctive iron-sulfur cluster where one iron atom is coordinated by two histidine residues rather than two cysteine residues, which is a defining feature of Rieske proteins . The mature protein functions within the mitochondrial membrane as part of the electron transport chain.

How does the structure of the Rieske iron-sulfur protein relate to its function?

The Rieske protein structure consists of two subdomains dominated by antiparallel β-structure with variable numbers of α-helices. The smaller "cluster-binding" subdomain contains the [2Fe-2S] cluster with a topology resembling an incomplete antiparallel β-barrel . This structural arrangement is critical for function as it:

• Positions the iron-sulfur cluster optimally for electron transfer

• Enables conformational changes during redox reactions

• Provides specific binding interfaces for interaction with ubiquinol/plastoquinol

• Facilitates electron transfer to cytochrome c1 or cytochrome f

The unique coordination of one iron atom by two histidine residues (rather than cysteines) gives Rieske proteins distinctive redox properties, with reduction potentials ranging from -150 to +400 mV depending on their biological context . This structural feature is essential for the protein's role in transferring electrons from ubiquinol to the cytochrome c1 component of the complex.

What consensus sequence characterizes the Rieske family of proteins?

The Rieske protein family is characterized by a highly conserved consensus sequence that coordinates the [2Fe-2S] cluster:

Cys-Xaa-His-(Xaa)15–17-Cys-Xaa-Xaa-His

In this motif:

• The first cysteine and histidine coordinate one iron atom

• The second cysteine and histidine coordinate the other iron atom

• The specific spacing between these coordination sites is critical for proper cluster formation

This sequence is evolutionarily conserved across various organisms, including plants, animals, and bacteria, indicating its fundamental importance to electron transfer functions in biological systems.

What expression systems are optimal for producing functional recombinant Nicotiana tabacum Rieske-2 protein?

For expressing recombinant Nicotiana tabacum Rieske-2 protein, researchers should consider:

Bacterial expression systems:

• E. coli BL21(DE3) with pET vector systems can be effective when expressing only the soluble domain (amino acids 61-272)

• Co-expression with iron-sulfur cluster assembly proteins (ISC) improves yield of properly folded protein

• Growth at lower temperatures (16-20°C) after induction enhances proper folding

Eukaryotic expression systems:

• Yeast (Pichia pastoris or Saccharomyces cerevisiae) systems provide better post-translational modifications

• Plant-based expression systems (tobacco BY-2 cells or Nicotiana benthamiana) offer native-like folding environment

Methodological considerations:

• Include specific protease inhibitors during purification to prevent degradation

• Maintain reducing conditions with DTT or β-mercaptoethanol to protect the [Fe-S] cluster

• Store in Tris-based buffer with 50% glycerol at -20°C to maintain stability

• Express only the mature protein (without mitochondrial targeting sequence) for improved solubility

• Consider adding a cleavable His-tag for affinity purification that can be removed to study native-like protein

How can researchers assess the integrity and activity of purified recombinant Rieske protein?

Spectroscopic characterization:

• UV-visible spectroscopy: Active Rieske proteins show characteristic absorption peaks at approximately 330, 458, and 560 nm

• Electron paramagnetic resonance (EPR): Provides information about the redox state of the [2Fe-2S] cluster

• Circular dichroism: Confirms proper secondary structure folding

Functional assays:

• Electron transfer activity can be measured using artificial electron donors (decylubiquinol) and acceptors (cytochrome c)

• Reduction potential determination by potentiometric titration

• Oxygen consumption measurements when incorporated into complex III

Structural verification:

• Mass spectrometry to confirm identity and modifications

• Limited proteolysis to assess proper folding

• Thermal shift assays to determine protein stability

What role does the Rieske protein play in plant responses to oxidative stress?

Plant Rieske proteins, including the Nicotiana tabacum Cytochrome b-c1 complex subunit Rieske-2, are critical components in the cellular response to oxidative stress:

Regulatory functions:

• The redox state of the Rieske protein influences respiratory chain activity under stress conditions

• Modifications to the Rieske protein can alter electron flow in mitochondria, affecting ROS production

• Plant Rieske proteins may participate in retrograde signaling from mitochondria to nucleus during stress

Experimental evidence from tobacco research:
Tobacco plants exposed to oxidative stressors (including tobacco smoke compounds) exhibit altered mitochondrial function partially mediated through changes in respiratory complex activity . Studies show that:

• Oxidative stress induces post-translational modifications of mitochondrial proteins

• Mitochondrial deacetylase Sirt3 expression decreases under oxidative stress conditions, leading to hyperacetylation of mitochondrial proteins

• SOD2 hyperacetylation is observed in response to oxidative stressors, potentially affecting mitochondrial redox balance

Methodological approaches to study this relationship:

• Gene silencing/knockout studies examining plant response to oxidative stressors

• Proteomic analysis of Rieske protein modifications under oxidative stress

• Metabolic flux analysis to determine changes in electron transport during stress response

• In vitro oxidation studies with purified recombinant protein

How can site-directed mutagenesis be applied to investigate the structure-function relationship of the Rieske iron-sulfur protein?

Site-directed mutagenesis is a powerful approach for investigating critical residues in the Rieske protein:

Key targets for mutagenesis:

Experimental workflow:

• Generate mutations using PCR-based methods or synthetic gene fragments

• Express and purify mutant proteins under identical conditions

• Characterize biophysical properties (stability, cluster incorporation)

• Measure functional parameters (redox potential, electron transfer rates)

• Determine structural changes using crystallography or spectroscopy

Case studies from related Rieske proteins:
Mutations in the conserved histidine ligands often result in complete loss of cluster assembly, while mutations in surrounding residues can fine-tune the redox potential, demonstrating the precise structural requirements for function.

What analytical methods are most effective for studying the interaction between the Rieske subunit and other components of the respiratory chain?

In vitro biochemical approaches:

• Co-immunoprecipitation with antibodies against the Rieske protein or interacting partners

• Surface plasmon resonance to measure binding kinetics

• Isothermal titration calorimetry for thermodynamic parameters of interactions

• Chemical cross-linking coupled with mass spectrometry to identify interaction interfaces

Structural methods:

• Cryo-electron microscopy of intact respiratory complexes

• X-ray crystallography of co-crystallized components

• Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• NMR studies of labeled domains to detect conformational changes upon interaction

Cellular approaches:

• FRET/BRET assays with fluorescently tagged proteins

• Proximity ligation assays in fixed cells or tissues

• Bimolecular fluorescence complementation in plant cells

Data analysis considerations:

• Control experiments must include non-interacting proteins of similar size/charge

• Multiple techniques should be employed to validate interactions

• Functional assays should accompany binding studies to confirm physiological relevance

• Native conditions should be maintained when possible to preserve relevant interactions

How can recombinant Nicotiana tabacum Rieske protein be used in comparative studies of plant respiratory systems?

The recombinant Nicotiana tabacum Rieske-2 protein serves as a valuable model for comparative studies across plant species due to its well-characterized properties. Researchers can use this protein to:

Comparative evolutionary analysis:

• Align sequences of Rieske proteins across plant species to identify conserved vs. variable regions

• Compare biochemical properties (redox potential, stability) of Rieske proteins from different plant lineages

• Investigate species-specific adaptations in respiratory chain components

Methodological approach:

• Express recombinant Rieske proteins from multiple plant species under identical conditions

• Perform side-by-side biochemical characterization

• Measure functional parameters in reconstituted systems

• Correlate differences to physiological adaptations or evolutionary distance

Applications in plant stress biology:
The Nicotiana tabacum Rieske-2 protein can serve as a reference for understanding how respiratory chain components adapt to different environmental conditions across plant species, particularly in response to oxidative stress conditions .

What techniques are most effective for studying post-translational modifications of the Rieske protein?

Post-translational modifications (PTMs) of Rieske proteins can significantly impact their function. Research shows that oxidative stress can induce modifications in mitochondrial proteins, including hyperacetylation . Effective techniques include:

Mass spectrometry approaches:

• Bottom-up proteomics with enrichment strategies for specific PTMs

• Top-down proteomics for intact protein analysis

• Targeted multiple reaction monitoring for quantitative analysis of specific modifications

Specific modification analysis:

• Phosphorylation: Phos-tag gels, phospho-specific antibodies, 32P labeling

• Acetylation: Anti-acetyllysine antibodies, HDAC inhibitor treatments

• Oxidative modifications: Redox proteomics, dimedone labeling for sulfenic acids

Functional correlation:

• Site-directed mutagenesis of modified residues to mimic or prevent modifications

• In vitro enzymatic modification systems

• Activity assays before and after specific modification events

Biological context:
Research indicates that mitochondrial proteins in tobacco are subject to acetylation regulated by Sirt3, and oxidative stress conditions can lead to hyperacetylation through reduced Sirt3 expression . These findings suggest that the Rieske protein may also be subject to acetylation-based regulation.

How do mutations in the Rieske protein affect plant bioenergetics and stress responses?

Mutations in the Rieske protein can have profound effects on plant energy metabolism and stress tolerance:

Bioenergetic impacts:

• Alterations in electron transfer efficiency can affect ATP production

• Changes in the reduction potential of the [2Fe-2S] cluster may shift electron flow distributions

• Structural mutations can impact supercomplex formation in mitochondrial membranes

Stress response implications:

• Some mutations may increase reactive oxygen species (ROS) production

• Altered Rieske function can affect mitochondrial retrograde signaling

• Changes in respiratory efficiency may impact plant tolerance to environmental stressors

Experimental approaches:

• Generate transgenic plants expressing mutated Rieske proteins

• Measure respiration rates, ATP production, and ROS generation

• Assess growth and development under normal and stress conditions

• Analyze transcriptional responses to identify affected pathways

Case study from related research:
Studies on tobacco plants exposed to cigarette smoke show increased mitochondrial oxidative stress and altered function of respiratory chain components . This suggests that understanding Rieske protein function could provide insights into plant responses to environmental toxicants.

What are the main challenges in maintaining stability of the recombinant Rieske protein during purification?

Rieske proteins present several technical challenges during purification due to their iron-sulfur cluster and membrane association:

Stability challenges:

• Oxygen sensitivity of the [2Fe-2S] cluster

• Tendency for aggregation when removed from membrane environment

• Proteolytic susceptibility of flexible regions

• Potential for disulfide bond formation with free cysteines

Methodological solutions:

• Perform all purification steps under low oxygen conditions (nitrogen atmosphere or glove box)

• Include reducing agents (DTT, β-mercaptoethanol) in all buffers

• Add glycerol (50%) to stabilize protein structure during storage

• Use protease inhibitor cocktails specifically designed for plant proteins

• Add mild detergents to mimic membrane environment

• Work at 4°C and minimize freeze-thaw cycles

• Consider using fusion tags that enhance solubility

Storage recommendations:
For optimal stability, store purified Nicotiana tabacum Rieske-2 protein in Tris-based buffer with 50% glycerol at -20°C for regular use, or at -80°C for long-term storage . Avoid repeated freezing and thawing by preparing small working aliquots to be kept at 4°C for up to one week.

How can researchers effectively design experiments to study the redox properties of the Rieske iron-sulfur cluster?

Experimental design considerations:

Methodological recommendations:

• Use multiple complementary techniques to cross-validate findings

• Include proper controls (denatured protein, known redox proteins)

• Carefully control experimental conditions (pH, ionic strength, temperature)

• Consider protein environment effects on redox properties

• Account for potential cooperative effects with other redox centers

Data analysis approach:

• Fit experimental data to appropriate electrochemical models

• Consider the number of electrons transferred in each step

• Account for protein concentration and active site accessibility

• Compare results across different experimental conditions to establish reproducibility

How does the Rieske protein contribute to respiratory chain supercomplexes and their regulation?

Recent research suggests that Rieske proteins play roles beyond electron transfer in the organization and regulation of respiratory chain supercomplexes:

Structural contributions:

• The Rieske domain's movement between b and c1 subunits may influence supercomplex stability

• Specific interactions between the Rieske protein and other complex components can serve as nucleation points for supercomplex assembly

• Post-translational modifications of the Rieske protein might regulate these interactions

Methodological approaches:

• Cryo-electron microscopy of intact supercomplexes with and without specific perturbations

• Crosslinking mass spectrometry to map interaction interfaces

• Mutagenesis of putative interaction sites followed by BN-PAGE analysis of supercomplex formation

• Computational modeling of supercomplex dynamics

Research questions at the frontier:

• Does the Rieske protein serve as a sensor for respiratory chain regulation?

• How do modifications of the Rieske protein influence respiratory chain organization?

• Can the Rieske protein be targeted to modulate respiratory efficiency in plants?

What is the relationship between mitochondrial oxidative stress, Rieske protein function, and plant cellular signaling?

The relationship between oxidative stress and Rieske protein function represents an important frontier in plant mitochondrial research:

Current understanding:
Research indicates that tobacco smoking induces mitochondrial oxidative stress and affects mitochondrial protein function through mechanisms including reduced expression of the mitochondrial deacetylase Sirt3 and hyperacetylation of mitochondrial proteins . These changes can alter electron transport chain function and increase reactive oxygen species production.

Key research questions:

• Does oxidative modification of the Rieske protein serve as a signal for mitochondrial stress?

• How do changes in Rieske protein function contribute to retrograde signaling?

• What role does the Rieske protein play in balancing energy production versus oxidative damage?

Experimental approaches:

• Site-specific oxidative modification of recombinant protein followed by functional analysis

• Development of redox-sensitive probes based on Rieske protein domains

• Identification of interacting partners under normal versus oxidative stress conditions

• Analysis of transcriptional responses to specific Rieske protein perturbations

What are the most promising research directions for Nicotiana tabacum Rieske protein studies?

Future research on the Nicotiana tabacum Cytochrome b-c1 complex subunit Rieske-2 should focus on:

• Detailed structure-function studies using advanced biophysical techniques

• Investigation of its role in plant stress responses, particularly oxidative stress

• Exploration of its potential as a target for enhancing plant resilience

• Comparative studies across plant species to understand evolutionary adaptations

• Analysis of its role in mitochondrial-chloroplast communication in plant cells

Research on tobacco Rieske proteins may provide valuable insights into how plants maintain energetic homeostasis under stress conditions, potentially leading to applications in crop improvement and biotechnology.

How can cross-disciplinary approaches enhance our understanding of Rieske protein biology?

The most significant advances in understanding Rieske protein biology will likely come from integrating multiple disciplines:

Integration of structural biology with plant physiology:
Correlating atomic-level structural details with whole-plant phenotypes

Combination of biochemistry with systems biology:
Understanding how Rieske protein properties influence metabolic networks

Merging evolutionary biology with biophysics:
Exploring how selective pressures have shaped Rieske protein properties across plant lineages

Computational approaches: Using molecular dynamics simulations and machine learning to predict structure-function relationships and identify critical determinants of Rieske protein function