Recombinant Synechocystis sp. Rubredoxin (rub)
Description
Recombinant Synechocystis sp. Rubredoxin (rub): Overview
Recombinant Synechocystis sp. Rubredoxin (rub) is a small, iron-sulfur protein critical for photosystem II (PSII) biogenesis and function in oxygenic phototrophs. It belongs to a conserved family of rubredoxins unique to cyanobacteria, algae, and plants, playing a pivotal role in electron transfer and redox regulation during photosynthesis .
Sequence Homology
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Shares 70% identity with rubredoxins in Chlamydomonas reinhardtii and Arabidopsis thaliana, underscoring evolutionary conservation .
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Diverges from archaeal and bacterial rubredoxins (e.g., Desulfovibrio vulgaris), which function in anaerobic processes .
Functional Role in Photosynthesis
RubA is indispensable for PSII activity, as evidenced by studies in Synechocystis mutants:
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ΔrubA Mutant Phenotype:
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Mechanism: Acts as a catalytic factor in D1/D2 heterodimer formation, not as a structural subunit .
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Redox Regulation: The RD domain senses oxidative changes, modulating PSII assembly under fluctuating light .
Knockout Experiments
| Strain | PSII Activity | D1/D2 Levels | Chlorophyll Fluorescence |
|---|---|---|---|
| Wild-Type | 100% | 100% | Normal |
| ΔrubA Mutant | ~70% | ~40% | Reduced |
| Overexpression | Restored | Restored | Wild-Type |
| Data from . |
Complementation
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Overexpression of rubA restores PSII activity, confirming its non-stoichiometric role .
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Co-expression with ycf48 (a PSII assembly factor) rescues PSII defects, highlighting functional synergy .
Metabolic Engineering
Recombinant Synechocystis strains engineered for rubredoxin overexpression may enhance:
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Photosynthetic Efficiency: By stabilizing PSII under oxidative stress .
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Biofuel Production: Altered electron transfer pathways could optimize metabolic flux toward biofuels like polyhydroxyalkanoates (PHA) .
Redox-Sensing Platforms
The RD domain’s redox responsiveness offers potential for:
Product Specs
Target Background
KEGG: syn:slr2033
STRING: 1148.SYNGTS_0515
Q&A
Basic Research Questions
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What is the fundamental role of rubredoxin in Synechocystis sp. and how does it function in photosynthesis?
Rubredoxin in Synechocystis sp. PCC 6803 (encoded by the rubA gene, also known as slr2033) is a small iron-sulfur protein with a unique domain architecture consisting of a rubredoxin domain fused to a C-terminal transmembrane helix that anchors it to the thylakoid membrane. Experimental evidence indicates that this protein is specifically required for Photosystem II (PSII) accumulation and function .
Interestingly, this finding contrasts with research on Synechococcus sp. PCC 7002, where rubA inactivation caused a loss of Photosystem I (PSI) activity while PSII remained at about 80% of wild-type levels . This discrepancy suggests potential species-specific functions for rubredoxin in different cyanobacteria.
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How is rubredoxin structurally distinct in oxygenic phototrophs compared to other organisms?
Rubredoxins in oxygenic phototrophs exhibit a unique domain architecture that distinguishes them from those found in other bacteria and archaea:
| Characteristic | Rubredoxin in Oxygenic Phototrophs | Canonical Rubredoxin in Other Organisms |
|---|---|---|
| Structure | Rubredoxin domain fused to C-terminal transmembrane helix | Usually soluble protein consisting almost entirely of rubredoxin domain |
| Localization | Anchored in thylakoid membrane | Cytoplasmic |
| Phylogeny | Forms a distinct clade in phylogenetic analyses | More diverse evolutionary relationships |
| Distribution | Present in all sequenced PSII-containing organisms | Found in various Archaea and bacteria |
Phylogenetic reconstruction shows that rubredoxins from PSII-containing organisms form a clade distinct from all others, suggesting that a membrane-bound rubredoxin was likely present in the most recent common ancestor of all oxygenic cyanobacteria and plastids . This membrane association is supported by detection of rubredoxin homologs in highly-purified thylakoid membranes from Synechococcus sp. PCC 7002 and in the thylakoid membrane proteome of Arabidopsis thaliana .
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What experimental approaches are used to investigate rubredoxin function in photosynthetic organisms?
Researchers employ multiple complementary techniques to study rubredoxin function:
| Technique | Methodological Details | Information Obtained |
|---|---|---|
| Gene Knockout | Replacing rubA with antibiotic resistance markers (e.g., kanamycin resistance nptI) and confirming segregation via PCR | Phenotypic effects of rubredoxin absence |
| Chlorophyll Fluorescence | Measuring variable fluorescence in wild-type and mutant strains under different growth conditions | Assessment of PSII activity |
| Immunoblot Analysis | Western blotting with antibodies against specific photosystem components (e.g., PsaA, PsaC, and PsaD for PSI; PSII subunits) | Quantification of photosystem protein levels |
| Spectroscopic Analysis | Measurements of ΔA520 nm and P700 redox changes | Evaluation of electron transport chain components |
| Complementation Studies | Transforming mutants with DNA fragments containing only the rubredoxin gene, its promoter, and 3′ UTR | Confirmation of rubredoxin's specific role |
Using these approaches, researchers demonstrated that without RBD1 (rubredoxin), the Chlamydomonas 2pac mutant does not accumulate PSII as assayed by chlorophyll a fluorescence, immunodetection of PSII subunits, and measurements of ΔA520 nm. PSII accumulation was restored via transformation with a small fragment of DNA containing only the RBD1 gene .
Advanced Research Questions
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How can researchers reconcile the contradictory findings regarding rubredoxin function in different cyanobacteria?
The contradictory findings between Synechocystis sp. PCC 6803 (where rubredoxin affects PSII) and Synechococcus sp. PCC 7002 (where it affects PSI) represent an intriguing research puzzle. Several investigative approaches can help reconcile these findings:
| Approach | Methodology | Research Value |
|---|---|---|
| Comparative Genomics | Analyzing genomic context of rubA between species; examining the conserved genomic location next to five genes involved in PSII function | May reveal species-specific adaptations |
| Cross-Species Complementation | Expressing Synechocystis rubredoxin in Synechococcus rubA mutants and vice versa | Determines if functional differences arise from the protein or cellular context |
| Protein-Protein Interaction Studies | Co-immunoprecipitation, yeast two-hybrid, or pull-down assays | Identifies different interaction partners in each species |
| Multiple Rubredoxin Analysis | Identifying and characterizing additional rubredoxin-like proteins | May reveal specialized functions for PSI vs. PSII assembly |
The search results note that in Chlamydomonas and Arabidopsis, there appear to be multiple chloroplast-localized rubredoxins, suggesting that different homologs might function in different aspects of photosystem assembly . This hypothesis could be tested by systematically characterizing each rubredoxin homolog's function.
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What methodologies can be used to investigate the redox properties of rubredoxin in Synechocystis?
The redox properties of rubredoxin are central to its biological function. Several sophisticated methodologies can elucidate these properties:
Research on other rubredoxin domains shows they can switch between a reduced, metal-bound folded state and an oxidized, metal-free unfolded state . These approaches could reveal whether Synechocystis rubredoxin undergoes similar transitions and how they relate to its function in photosystem assembly.
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What are the optimal expression and purification methods for recombinant Synechocystis rubredoxin?
Based on research with other rubredoxins, the following methods are recommended:
The "RubyTag" approach (using rubredoxin from Thermotoga maritima as a colored fusion tag) demonstrates that rubredoxin's spectroscopic properties can be leveraged for monitoring expression and purification . This approach could be adapted for Synechocystis rubredoxin, potentially enhancing yield and purity.
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How can site-directed mutagenesis illuminate the structure-function relationship of Synechocystis rubredoxin?
Site-directed mutagenesis provides powerful insights into rubredoxin's functional mechanisms:
Studies with other rubredoxin domains show that replacing the cysteines with alanines or serines disables redox regulation and results in significant structural changes as detected by fluorescence and NMR spectroscopy . Similar approaches with Synechocystis rubredoxin could reveal how specific residues contribute to its role in photosystem assembly.
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What are the implications of rubredoxin's evolutionary conservation for understanding oxygenic photosynthesis development?
The evolutionary profile of rubredoxin provides critical insights into photosynthesis development:
The search results explicitly state that "rubredoxin was likely important in the evolution of oxygenic photosynthesis" . Its specific requirement for PSII activity across diverse photosynthetic lineages suggests it was an early innovation in the development of water-splitting photosynthesis.
Interestingly, rubredoxin appears to function catalytically rather than as a stoichiometric subunit of PSII, as PSII activity is fully restored in complemented lines despite relatively low levels of rubredoxin protein accumulation . This suggests it has a regulatory or assembly role that has been maintained throughout evolution.
