Recombinant Nicotiana tabacum Cytochrome b-c1 complex subunit Rieske-4, mitochondrial

What is the Rieske protein and what distinguishes it from other iron-sulfur proteins?

Rieske proteins are specialized iron-sulfur protein components of cytochrome bc1 complexes (Complex III) in the mitochondrial respiratory chain. They contain a unique [2Fe-2S] cluster where one iron atom is coordinated by two histidine residues rather than the typical two cysteine residues seen in other iron-sulfur proteins . This distinctive coordination gives Rieske proteins their characteristic redox properties with reduction potentials ranging from -150 to +400 mV . They play a critical role in electron transfer during oxidative phosphorylation, accepting electrons from ubiquinol and transferring them to cytochrome c1, which contributes to the electrochemical potential difference used for ATP synthesis .

The Rieske subunit functions by binding ubiquinol anions, transferring electrons to the [2Fe-2S] cluster, and then releasing the electrons to cytochrome c or cytochrome f heme iron . Notably, the reduction of the Rieske center increases its affinity for substrates by several orders of magnitude, which stabilizes the semiquinone radical at the Q(P) site .

How do Rieske-1 and Rieske-4 isoforms in Nicotiana tabacum differ structurally?

The Nicotiana tabacum (tobacco) mitochondrial genome encodes multiple Rieske protein isoforms with distinct structural characteristics. Comparing the two recombinant proteins reveals several key differences:

While both proteins share high sequence similarity (as evident from the sequences provided in search results ), they exhibit distinct amino acid variations that may influence their functional properties and interactions within the respiratory complex .

What role does the Rieske protein play in respiratory and photosynthetic electron transport chains?

Rieske proteins serve critical functions in both respiratory and photosynthetic electron transport chains:

In mitochondria, the Rieske protein is an essential component of the cytochrome bc1 complex (Complex III), which catalyzes the oxidation-reduction reaction between ubiquinol and cytochrome c . This electron transfer contributes to the proton gradient across the inner mitochondrial membrane that drives ATP synthesis .

In photosynthetic organisms, a similar protein functions in the cytochrome b6f complex of chloroplasts, facilitating electron transfer from plastoquinol to plastocyanin . Recent research has demonstrated that overexpression of the Rieske FeS protein in tobacco leads to up to a 40% increase in the abundance of the cytochrome b6f complex, accompanied by enhanced cytochrome f activity . Analysis of these transgenic plants revealed a more oxidized primary quinone acceptor of photosystem II and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance .

Additionally, these plants showed faster establishment of the energy-dependent component of nonphotochemical quenching (qE), suggesting a more rapid buildup of the transmembrane proton gradient . These findings indicate that the Rieske protein plays a rate-limiting role in photosynthetic electron transport.

What expression systems are optimal for producing functional recombinant Rieske proteins?

Based on current research protocols, E. coli represents the predominant expression system for recombinant Nicotiana tabacum Rieske proteins. The published data indicates successful expression of both Rieske-1 and Rieske-4 proteins in E. coli with N-terminal His tags . The bacterial expression approach allows for high yield production while maintaining the functional integrity of the protein's [2Fe-2S] cluster.

For researchers seeking alternative expression systems, particularly when post-translational modifications are critical, yeast expression systems show promise. For instance, Saccharomyces cerevisiae strain WAT11, which contains Arabidopsis thaliana P450 reductase necessary for catalytic activity of cytochrome enzymes, has been successfully used for expressing related proteins . This system might be advantageous when studying protein-protein interactions within the complex.

For mitochondrial expression studies, biolistic transformation has been demonstrated as an effective approach for relocating nuclear Rieske genes into mitochondria. In one study, researchers successfully integrated and expressed a mitochondrially encoded copy of the Rieske gene (RIP1m) downstream of the cox1 gene by fusing it with appropriate regulatory elements .

What purification strategies yield the highest activity for His-tagged Rieske proteins?

For optimal purification of His-tagged recombinant Rieske proteins, the following methodological approach is recommended based on current protocols:

• Initial preparation: Brief centrifugation of the protein vial prior to opening to bring contents to the bottom .

• Immobilized metal affinity chromatography (IMAC): Standard Ni-NTA purification with optimized imidazole gradients to ensure high purity (>90% as determined by SDS-PAGE) .

• Reconstitution conditions: Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Buffer composition: Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been demonstrated to maintain protein stability during and after purification .

• Storage stabilization: Addition of 5-50% glycerol (final concentration) for long-term storage, with 50% being the recommended default concentration .

It's critical to note that repeated freeze-thaw cycles significantly impair protein activity. Therefore, preparing working aliquots stored at 4°C for up to one week, while maintaining the main stock at -20°C/-80°C, is strongly advised .

How can the functional integrity of the [2Fe-2S] cluster be verified after purification?

Verifying the functional integrity of the [2Fe-2S] cluster in purified Rieske proteins requires a multi-analytical approach:

• Spectroscopic analysis: UV-visible spectroscopy can reveal characteristic absorption patterns of intact [2Fe-2S] clusters. Reduced and oxidized forms of the cluster show distinctive spectral signatures that confirm cluster integrity.

• Activity assays: Functional assessment through electron transfer activity measurements provides direct evidence of intact [2Fe-2S] clusters. Research on Rieske FeS overexpression in tobacco demonstrates that enhanced cytochrome f activity correlates with functional cluster integration .

• EPR spectroscopy: Electron paramagnetic resonance can detect the paramagnetic properties of the [2Fe-2S] cluster in its reduced state, providing detailed information about cluster environment and redox state.

• Mutagenesis validation: Studies with mutants having lesions in the [2Fe-2S] cluster binding site have demonstrated loss of function, confirming the essential nature of this cluster for protein activity . Comparisons between wild-type and mutant proteins can validate cluster integrity in recombinant preparations.

The importance of the [2Fe-2S] cluster is underscored by research showing that mutations in this binding site eliminated the ability of Rieske-type proteins to induce changes in mitochondrial morphology and growth arrest .

What techniques are most effective for assessing electron transfer activity of recombinant Rieske proteins?

Electron transfer activity of recombinant Rieske proteins can be assessed using several complementary approaches:

• Light-induced fluorescence transient (LIFT) technique: This method has successfully revealed differences in electron transport capabilities between wild-type and Rieske FeS-overexpressing plants. The technique can detect a more oxidized primary quinone acceptor of photosystem II (QA) and plastoquinone pool, as well as faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance .

• Cytochrome f activity assays: In vitro measurement of cytochrome f activity provides direct evidence of functional electron transfer. Enhanced activity has been documented in preparations containing overexpressed Rieske FeS protein, indicating successful integration into functional complexes .

• Nonphotochemical quenching (NPQ) analysis: Measurements of qE (the energy-dependent component of NPQ) establishment rates can indirectly assess electron transport. Faster qE establishment suggests more rapid buildup of transmembrane proton gradients, supporting increased cytochrome b6f activity .

• Complementation studies: Functional complementation, as demonstrated in yeast studies where mitochondrially encoded Rieske genes rescued nuclear gene deletions, provides compelling evidence of electron transfer capacity .

When designing electron transfer assays, researchers should consider both steady-state measurements and dynamic responses to changing conditions, as transient increases in activity may not always translate to sustained enhancement of steady-state electron transport rates .

How does overexpression of Rieske proteins impact photosynthetic efficiency in tobacco?

Research on Rieske FeS protein overexpression in Nicotiana tabacum has revealed complex effects on photosynthetic efficiency:

Overexpression led to up to a 40% increase in cytochrome b6f complex abundance accompanied by enhanced in vitro cytochrome f activity, demonstrating successful integration of the recombinant protein into functional complexes . Transgenic plants exhibited several promising physiological changes, including:

• More oxidized primary quinone acceptor of photosystem II (QA) and plastoquinone pool

• Faster electron transport from the plastoquinone pool to photosystem I upon light intensity changes

• More rapid establishment of energy-dependent nonphotochemical quenching (qE)

These findings indicate that while Rieske protein overexpression enhances the abundance of functional cytochrome b6f, it may need to be combined with other traits to achieve sustained improvements in photosynthetic efficiency and plant productivity .

What role does the [2Fe-2S] cluster binding site play in Rieske protein function?

The [2Fe-2S] cluster binding site is essential for Rieske protein function, as demonstrated by multiple studies:

A study examining a novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript found that nuclear localization of AIFL-I4 induced changes in mitochondrial morphology and suppressed cell proliferation . Critically, a mutant variant with a lesion in the [2Fe-2S] cluster binding site failed to induce these phenotypes, confirming that the iron-sulfur cluster is essential for the protein's biological activity .

The [2Fe-2S] cluster in Rieske proteins has unique coordination where one iron atom is coordinated by two histidine residues instead of cysteine residues . This distinct coordination pattern contributes to the protein's unique redox properties and electron transfer capabilities. The reduction of the Rieske center increases its affinity for substrates by several orders of magnitude, stabilizing semiquinone radicals at the Q(P) site during electron transfer .

The highly conserved nature of the C-terminal region that binds the [2Fe-2S] cluster across diverse organisms underscores its fundamental importance in protein function . Modifications to this region through site-directed mutagenesis consistently disrupt electron transfer capabilities, providing further evidence of its critical role.

How does the Rieske protein integrate into the cytochrome b-c1 complex?

The integration of Rieske proteins into the cytochrome b-c1 complex involves specific structural domains and interaction interfaces:

The cytochrome b-c1 complex (Complex III) contains 11 subunits in total: 3 respiratory subunits (cytochrome B, cytochrome C1, Rieske protein), 2 core proteins, and 6 low-molecular weight proteins . The Rieske protein occupies a strategic position within this multiprotein assembly.

Rieske proteins contain a transmembrane domain that anchors them in the membrane. The "Cytochrome b-c1 complex subunit Rieske, transmembrane domain" is recognized as a distinct structural motif (Pfam: PF02921) . This domain is critical for proper positioning of the protein within the complex.

During electron transfer, the Rieske subunit undergoes conformational changes. It accepts electrons from cytochrome b and then undergoes a conformational shift to attach to cytochrome c1, where the electron is transferred to the heme carried by cytochrome c1 . This electron is subsequently transferred to cytochrome c, creating a reduced species that separates from the complex and moves to cytochrome c oxidase (Complex IV) .

Studies relocating the nuclear Rieske gene into mitochondria have shown that proper integration requires appropriate targeting sequences and interactions with other complex components . The ability of mitochondrially encoded Rieske proteins to complement nuclear gene deletions confirms that the protein can be successfully incorporated into functional complexes regardless of its genetic origin .

What is known about the evolution of Rieske proteins across different species?

Rieske proteins demonstrate remarkable evolutionary conservation across diverse organisms while exhibiting adaptations to specific metabolic requirements:

The Rieske iron-sulfur proteins are found throughout the tree of life, appearing in plants, animals, and bacteria with electron reduction potentials ranging from -150 to +400 mV . This range reflects adaptations to different cellular environments and metabolic needs.

Phylogenetic analysis reveals significant conservation of the Rieske protein structure, particularly in the C-terminal region that binds the [2Fe-2S] cluster . The human ortholog of the Rieske protein, UQCRFS1 (Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1), functions similarly in the respiratory chain, underscoring evolutionary conservation .

In photosynthetic organisms, Rieske proteins have been adapted to function in both mitochondrial and chloroplast electron transport chains. In chloroplasts, they are components of the cytochrome b6f complex, which is functionally analogous to the mitochondrial cytochrome bc1 complex . This dual role highlights the protein's evolutionary versatility.

Tobacco (Nicotiana tabacum) possesses multiple Rieske protein isoforms, including Rieske-1 (P49729) and Rieske-4 (P51134) , suggesting gene duplication events and potential functional specialization. Other plants like Malus domestica (apple) also contain cytochrome b-c1 complex subunit Rieske proteins with high sequence similarity to those in tobacco .

How can site-directed mutagenesis of Rieske proteins enhance our understanding of electron transfer mechanisms?

Site-directed mutagenesis offers powerful insights into the mechanistic details of Rieske protein function in electron transfer:

Strategic mutations targeting the [2Fe-2S] cluster coordination sites can reveal the contribution of specific residues to redox properties. Research with Rieske-type proteins has already demonstrated that mutations in the [2Fe-2S] cluster binding site eliminate biological effects such as changes in mitochondrial morphology and growth suppression . This confirms the critical nature of these residues for electron transfer function.

Mutations affecting the interface between the Rieske protein and other components of the cytochrome bc1 complex can elucidate protein-protein interactions essential for efficient electron transfer. By systematically altering residues at these interfaces, researchers can map the communication pathways within the complex.

The unique coordination of one iron atom by two histidine residues in Rieske proteins contributes to their distinctive redox properties . Mutations converting these histidines to cysteines (the more typical coordination in iron-sulfur proteins) could provide insights into the evolutionary advantages of the histidine coordination in electron transport chains.

Rieske proteins demonstrate enantioselectivity in their interactions with substrates. For example, after incubation with a synthetic racemic mixture of 9,10-epoxystearic acid, the residual epoxide showed a 40/60 ratio in favor of the 9R,10S enantiomer . Mutations affecting this selectivity could reveal structural determinants of substrate recognition.

What approaches can optimize Rieske protein overexpression for enhancing photosynthetic efficiency?

Several strategic approaches can optimize Rieske protein overexpression to enhance photosynthetic efficiency:

• Targeted expression strategies: While constitutive overexpression of Rieske FeS protein in tobacco increased complex abundance by up to 40%, it did not consistently enhance steady-state photosynthesis rates . Alternative expression strategies could include:

  • Tissue-specific promoters targeting photosynthetically active tissues

  • Light-responsive promoters to synchronize expression with photosynthetic demand

  • Stress-responsive elements to enhance expression under limiting conditions

• Multi-component engineering: Research suggests that Rieske FeS overexpression "may have the potential to increase plant productivity if combined with other traits" . Complementary approaches might include:

  • Co-expression with other rate-limiting components of the electron transport chain

  • Simultaneous enhancement of carbon fixation capacity through RuBisCO engineering

  • Optimization of photoprotection mechanisms to prevent photodamage during enhanced electron flow

• Stability and assembly optimization: Ensuring proper assembly of the cytochrome b6f complex is critical. Approaches could include:

  • Co-expression of chaperones that facilitate complex assembly

  • Engineering of post-translational modifications that enhance stability

  • Optimization of iron-sulfur cluster biogenesis to ensure full functionality

• Field validation protocols: Research has shown differences between laboratory and field performance . Comprehensive field testing should include:

  • Evaluation under fluctuating light conditions

  • Assessment across multiple growing seasons

  • Analysis of yield components and harvest index to determine translational impact

Current research indicates that while overexpression enhances the abundance of functional cytochrome b6f, the in vivo activity appears transiently increased only upon changes in irradiance . This suggests that dynamic light conditions may be where enhanced Rieske protein expression provides the greatest benefit.

What research directions could exploit the role of Rieske proteins in cellular signaling beyond electron transport?

Emerging evidence suggests Rieske proteins may have functions beyond their classical role in electron transport, opening several promising research directions:

• Mitochondrial morphology regulation: A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with retained intron 4 (AIFL-I4) has been shown to induce changes in mitochondrial morphology when localized to the nucleus . This suggests Rieske proteins may participate in retrograde signaling from mitochondria to the nucleus. Future research could explore:

  • The mechanisms by which Rieske proteins influence organelle morphology

  • The signaling pathways activated by nuclear-localized Rieske proteins

  • The evolutionary conservation of this signaling function across species

• Cell proliferation control: AIFL-I4 has been demonstrated to suppress cell proliferation, with this function dependent on an intact [2Fe-2S] cluster binding site . This suggests Rieske proteins may influence cell cycle regulation. Potential research directions include:

  • Identifying downstream effectors mediating growth suppression

  • Exploring therapeutic applications in hyperproliferative disorders

  • Investigating the relationship between metabolic status and cell cycle control via Rieske proteins

• Stress adaptation mechanisms: The role of Rieske proteins in electron transfer makes them potential sensors of cellular redox status. Future research could investigate:

  • How Rieske protein function changes under oxidative stress

  • Whether post-translational modifications of Rieske proteins serve as stress signals

  • The potential role of Rieske proteins in priming stress responses

• Synthetic biology applications: The unique properties of Rieske proteins could be exploited for novel biotechnological applications:

  • Design of synthetic redox sensors with customized response ranges

  • Creation of artificial electron transport chains with enhanced efficiency

  • Development of optogenetic tools leveraging the redox-sensitive nature of Rieske proteins

These research directions extend beyond the traditional focus on Rieske proteins in bioenergetics, potentially revealing novel functions with implications for cellular regulation, stress responses, and biotechnological applications.

What factors most commonly affect the stability of recombinant Rieske proteins?

Several critical factors influence the stability of recombinant Rieske proteins:

• Temperature sensitivity: Both Rieske-1 and Rieske-4 recombinant proteins require storage at -20°C/-80°C for long-term stability, with working aliquots maintained at 4°C for up to one week . This temperature dependence reflects the sensitivity of the [2Fe-2S] cluster to thermal denaturation.

• Freeze-thaw degradation: Multiple freeze-thaw cycles significantly impair protein integrity. Product documentation explicitly warns that "repeated freezing and thawing is not recommended" . This degradation likely results from ice crystal formation disrupting protein structure and potentially damaging the [2Fe-2S] cluster.

• Buffer composition effects: The recommended storage buffer (Tris/PBS-based buffer with 6% Trehalose, pH 8.0) has been optimized for Rieske protein stability . The slightly alkaline pH and inclusion of trehalose as an osmolyte help maintain native protein conformation and protect against denaturation.

• Cryoprotectant requirements: Addition of glycerol at 5-50% final concentration is recommended for long-term storage, with 50% being the default concentration . Glycerol prevents ice crystal formation and maintains protein in a more flexible state during freezing.

• Concentration considerations: Reconstitution to 0.1-1.0 mg/mL is recommended, suggesting this concentration range optimizes stability while preventing aggregation . Proteins at very high or very low concentrations often exhibit reduced stability.

• Oxidation susceptibility: The iron-sulfur cluster is sensitive to oxidation, which can disrupt electron transfer capability. While not explicitly mentioned in the search results, maintaining reducing conditions during handling may enhance stability.

How can researchers troubleshoot expression systems that yield low amounts of functional Rieske protein?

When troubleshooting low yields of functional recombinant Rieske proteins, researchers should consider these methodological approaches:

• Optimize codon usage: Adapt codons to the expression host's preference, particularly for rare codons that might limit translation efficiency. While E. coli is commonly used for expressing Nicotiana tabacum Rieske proteins , codon optimization can significantly enhance yields.

• Engineer expression constructs: Consider using different fusion tags beyond the standard His-tag. While His-tags are effective for purification , alternative tags like MBP (maltose-binding protein) can enhance solubility and proper folding.

• Adjust induction conditions: Optimize temperature, inducer concentration, and induction timing. Lower temperatures (16-20°C) during induction often improve folding of complex proteins containing cofactors like the [2Fe-2S] cluster.

• Supplement growth media: Add iron and sulfur sources to ensure adequate materials for [2Fe-2S] cluster assembly. Consider co-expression with iron-sulfur cluster assembly proteins to enhance proper cofactor incorporation.

• Explore alternative expression systems: While E. coli is commonly used , consider yeast systems like S. cerevisiae strain WAT11, which contains the necessary P450 reductase for proper electron transfer protein function .

• Implement in vivo complementation testing: Evaluate functionality through complementation assays, similar to studies where mitochondrially encoded Rieske genes rescued nuclear gene deletions . This approach verifies not just expression but functional integration.

• Assess protein solubility: Analyze both soluble and insoluble fractions to determine if the protein is being produced but forming inclusion bodies. Modify solubilization protocols if the protein is predominantly in the insoluble fraction.

What analytical methods can verify the quality and functionality of purified Rieske proteins?

Multiple analytical methods can verify the quality and functionality of purified Rieske proteins:

In combination, these methods provide comprehensive quality verification of purified recombinant Rieske proteins for research applications.