Recombinant Arabidopsis thaliana Cytochrome c oxidase subunit 2 (COX2)
Description
Production and Purification Methods
Recombinant COX2 is produced using heterologous expression systems:
Role in Complex IV Assembly
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COX2 interacts with the translocase OXA2b during membrane insertion. The C-terminal TPR domain of OXA2b binds nascent COX2, ensuring proper orientation and stability .
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Deletion of OXA2b’s TPR domain disrupts COX2 integration, leading to Complex IV deficiency and severe growth retardation in Arabidopsis .
Stress Response Coordination
While COX2 primarily functions in electron transport, its expression is indirectly linked to stress adaptation through crosstalk with alternative oxidases (AOX). For example:
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AOX1A upregulation under stress compensates for COX pathway inefficiencies, minimizing ROS production .
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COX2-deficient plants show heightened sensitivity to light and drought, emphasizing its role in maintaining redox homeostasis .
Research Applications
Recombinant COX2 is utilized in:
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Enzyme Kinetics: Studying electron transfer rates under varying oxygen tensions.
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Protein-Protein Interaction Assays: Mapping binding interfaces with OXA2b and cytochrome c .
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Structural Biology: Cryo-EM studies to resolve conformational changes during catalysis .
Challenges and Future Directions
Product Specs
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Target Background
Cytochrome c oxidase subunit 2 (COX2) is a crucial component of cytochrome c oxidase (Complex IV), the terminal enzyme in the mitochondrial electron transport chain. This enzyme drives oxidative phosphorylation, a process vital for cellular energy production. The respiratory chain comprises three multi-subunit complexes: succinate dehydrogenase (Complex II), ubiquinol-cytochrome c oxidoreductase (Complex III), and cytochrome c oxidase (Complex IV). These complexes work together to transfer electrons from NADH and succinate to molecular oxygen, generating an electrochemical gradient across the inner mitochondrial membrane. This gradient powers ATP synthesis and transmembrane transport. COX2, specifically, catalyzes the reduction of oxygen to water. Electrons from reduced cytochrome c in the intermembrane space are transferred through the copper A center (CuA) of subunit 2 and heme A of subunit 1 to the active site (a binuclear center, BNC, consisting of heme A3 and copper B (CuB)) in subunit 1. The BNC utilizes four electrons from cytochrome c and four protons from the mitochondrial matrix to reduce molecular oxygen to two water molecules.
KEGG: ath:ArthMp015
STRING: 3702.ATMG00160.1
Q&A
What experimental approaches are used to study COX2 assembly in Arabidopsis?
Basic Question
To investigate COX2 assembly, researchers employ co-immunoprecipitation (co-IP), pull-down assays, and firefly luciferase complementation imaging (LCI). These methods confirm direct interactions between COX2 and chaperones like mtHSC70-1 . For example:
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Co-IP: mtHSC70-1-GFP fusion proteins are immunoprecipitated using GFP-Trap, followed by immunoblotting with anti-Cox2 antibodies to detect interactions .
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Pull-down assays: GST-tagged mtHSC70-1 is incubated with MBP-tagged COX2, and binding is confirmed via SDS-PAGE and immunoblotting .
Advanced Question
For functional validation, blue native-PAGE (BN-PAGE) and COX activity assays are critical. BN-PAGE resolves mitochondrial complexes to assess COX2 incorporation into the COX holoenzyme . COX activity is quantified using Clark electrodes with cytochrome c as a substrate .
How does mtHSC70-1 influence COX2 stability and assembly?
Basic Question
mtHSC70-1 acts as a chaperone for COX2, ensuring its proper folding and integration into the COX complex. In mtHSC70-1 knockout mutants, COX2 levels in the COX complex are reduced, though total COX2 protein remains unchanged . This suggests mtHSC70-1 stabilizes COX2 within the complex rather than regulating its synthesis.
What are the key differences between COX2 and other cytochrome c oxidase subunits in Arabidopsis?
Basic Question
COX2 is a core subunit of the COX complex, whereas COX1 (mitochondrially encoded) and COX3 (nuclear-encoded) are also essential. COX2 interacts with mtHSC70-1 for proper assembly, unlike COX1, which does not bind mtHSC70-1 .
Advanced Question
COX11, a mitochondrial chaperone, is indispensable for copper insertion into COX2 and COX1 . In contrast, HCC1 (a copper chaperone) is critical for COX function, while HCC2 impacts UV-B stress responses but not COX activity .
How do cytonuclear interactions influence COX2 function in Arabidopsis?
Basic Question
COX2 is nuclear-encoded, but its proper assembly requires coordination with mitochondrial-encoded subunits (e.g., COX1) and chaperones like mtHSC70-1 and COX11. Disruptions in these interactions (e.g., via cytoplasmic exchanges in cytolines) can lead to compromised respiration and altered phenotypes .
Advanced Question
Intraspecific cytolines reveal that cytoplasmic diversity (e.g., mitochondrial genomes) interacts with nuclear-encoded COX2 to affect traits like seedling vigor and stress tolerance . This underscores the need to study COX2 in context-specific genetic backgrounds.
What methods validate recombinant COX2 functionality?
Basic Question
Recombinant COX2 is validated via Western blotting (to confirm protein expression) and immunoprecipitation (to assess interactions with chaperones like mtHSC70-1) .
Advanced Question
Enzyme activity assays: Recombinant COX2 is reconstituted into lipid bilayers or mitochondrial membranes, and COX activity is measured using substrates like reduced cytochrome c . BN-PAGE further verifies its incorporation into the COX complex .
How do promoter elements regulate COX2 expression in Arabidopsis?
Basic Question
COX2 expression is indirectly regulated by promoters of upstream genes (e.g., Cytc-1 and Cytc-2). The Cytc-1 promoter contains site II elements (TGGGCC/T) and internal telomeric repeats (AAACCCTAA), which bind TCP-domain transcription factors to drive expression in proliferating tissues (e.g., meristems) .
Advanced Question
Mutagenesis of site II elements abolishes Cytc-1 expression, while downstream telomeric repeats modulate transcriptional activity . These elements may coordinate expression of other COX-related genes, linking cell proliferation to mitochondrial respiration .
What challenges exist in studying COX2 in recombinant systems?
Basic Question
COX2 requires copper insertion (via COX11/HCC1) and chaperone assistance (mtHSC70-1) for proper folding. Recombinant systems must replicate these conditions, often using E. coli or insect cells with heterologous copper chaperones .
Advanced Question
Conformational instability: COX2 may misfold in non-native systems, necessitating in vitro refolding or mitochondrial import assays to assess functionality. Oxidative stress during expression can further compromise yield and activity .
How do HCC1 and HCC2 differ in their roles in COX2 biogenesis?
Basic Question
HCC1 is essential for COX2 function, likely delivering copper to the catalytic center, while HCC2 is dispensable for COX activity but enhances UV-B stress tolerance .
Advanced Question
COX activity assays show ~60% reduction in HCC1/ hcc1 mutants but no significant change in hcc2 knockouts . BN-PAGE reveals supercomplex associations in wild-type and hcc2 mutants, suggesting HCC2 may stabilize COX interactions with other respiratory complexes .
What are the implications of COX2 dysfunction for plant development?
Basic Question
COX2 defects impair mitochondrial respiration, leading to growth arrest in mtHSC70-1 mutants and embryo lethality in HCC1 knockouts .
Advanced Question
Pollen viability assays: hcc2 mutants show reduced UV-B tolerance, while HCC1 disruption causes sterility due to defective COX function . These phenotypes highlight COX2’s role in stress adaptation and reproductive success.
How is COX2 purified and characterized in recombinant systems?
Basic Question
COX2 is purified via affinity chromatography (e.g., His-tag or GST-tag systems) and size-exclusion chromatography to isolate monomeric/dimeric forms .
Advanced Question
Crystallography: Recombinant COX2 is co-expressed with mtHSC70-1 to stabilize its structure for X-ray crystallography . Electron microscopy resolves interactions with chaperones in near-native environments .
