Recombinant Saccharomyces cerevisiae Cytochrome c oxidase subunit 2 (COX2)

Basic Research Questions

• What is the localization and topology of Cox2p in Saccharomyces cerevisiae?

  Cox2p is localized in the mitochondrial inner membrane with its conserved cysteines in a Cx2C motif facing the intermembrane space . This topological arrangement is critical for proper function in electron transfer. The protein contains transmembrane domains that anchor it to the membrane while positioning its functional domains correctly for interaction with other proteins and cofactors. The orientation with conserved cysteines in the intermembrane space is particularly important as it allows for proper copper incorporation and interaction with copper chaperones. To study Cox2p localization, researchers typically employ subcellular fractionation, immunofluorescence microscopy with tagged variants, and protease protection assays to determine membrane orientation and topology.

• What are the main functions of Cox2p in yeast cytochrome c oxidase?

  Cox2p serves several critical functions in the cytochrome c oxidase (COX) complex of S. cerevisiae. Its primary role is harboring the binuclear CuA center, which receives electrons from cytochrome c and transfers them to the catalytic center in Cox1p . This electron transfer is essential for the reduction of molecular oxygen to water, coupled with proton translocation across the inner membrane. Cox2p also contributes to the structural integrity of the entire COX complex and influences the formation of respiratory supercomplexes . Research has demonstrated that Cox2p is absolutely essential for COX activity, as the enzyme does not assemble properly in the absence of functional Cox2p . The copper centers in Cox2p are particularly important for its electron transfer function, making proper metalation a critical aspect of Cox2p maturation.

• How is Cox2p processed and inserted into the mitochondrial membrane?

  The maturation and membrane insertion of Cox2p involve a coordinated process requiring specialized proteins. Cox2p is initially synthesized as a precursor with an N-terminal leader sequence. Cox18p and Cox20p play crucial roles in processing and membrane insertion of the Cox2p precursor . Cox20p functions as a chaperone for newly synthesized Cox2p, protecting it from degradation and facilitating its maturation . Cox18p is specifically involved in the export of the C-terminal domain of Cox2p across the inner membrane.

  The experimental approach to study Cox2p processing typically involves pulse-chase labeling of mitochondrial translation products, analysis of precursor processing in wildtype and mutant strains, and co-immunoprecipitation studies to identify interacting partners. Research has shown that in the absence of these processing factors, Cox2p fails to mature properly, leading to respiratory deficiencies. The double mutant cox20 dmo2 shows a reduced frequency of reversion to respiratory competency compared to the single cox20 mutant, indicating the importance of both factors in Cox2p processing .

• What key structural features of Cox2p are essential for its function?

  Several structural elements in Cox2p are critical for its proper function:

  Structural Feature	Function	Research Method
  Cx2C motif	Copper coordination and redox activity	Site-directed mutagenesis; functional assays
  Transmembrane helices	Membrane anchoring and complex stability	Hydrophobicity analysis; topology mapping
  CuA binding domain	Electron reception from cytochrome c	Metal binding assays; spectroscopy
  Intermembrane space domain	Interaction with cytochrome c	Crosslinking; co-immunoprecipitation

  The conserved cysteines in Cox2p are particularly important, as demonstrated by studies on similar motifs in interacting proteins where mutation of cysteine residues results in respiratory deficiency . These cysteines are likely involved in copper coordination and electron transfer functions. The proper folding and orientation of Cox2p's domains are essential for positioning the CuA center correctly for electron reception from cytochrome c and subsequent transfer to other components of the respiratory chain.

• How can researchers express and purify recombinant Cox2p for functional studies?

  Expression and purification of recombinant Cox2p for functional studies involves specialized techniques due to its membrane localization and mitochondrial encoding. Research has successfully employed genetic modification approaches where COX2 is fused to tags such as hemagglutinin (HA) or tandem HA followed by protein C tag (HAC) . These tagged versions are then expressed by substituting the fusion genes (COX2-HA and COX2-HAC) for the cox2 null allele in S. cerevisiae .

  For purification, affinity chromatography using antibody beads specific to the protein tag has proven effective . Mitochondria from cells expressing the tagged Cox2p are typically extracted with detergents like lauryl maltoside and purified on protein C antibody beads. The purity and identity of the isolated protein can be verified by SDS-PAGE, silver staining, and Western blotting . Importantly, research has shown that tagged versions of Cox2p retain functionality, although the HAC tag reduces COX assembly and activity by approximately 20% . This relatively minor impairment still makes tagged Cox2p suitable for most functional studies.