Recombinant Cyberlindnera mrakii Cytochrome c oxidase subunit 2 (COX2)

What is Cyberlindnera mrakii and why is its COX2 protein studied?

Cyberlindnera mrakii is a yeast species belonging to the Saccharomycotina subphylum. It is one of the many yeast species whose mitochondrial genomes have been sequenced and analyzed as part of broader studies on mitochondrial genome diversity . COX2 (Cytochrome c oxidase subunit 2) from C. mrakii is studied because it encodes a highly conserved protein that plays a crucial role in the electron transport chain of cellular respiration. Specifically, COX2 is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX), which is essential for ATP production .

The study of COX2 from diverse species like C. mrakii provides valuable insights into mitochondrial evolution, adaptation mechanisms, and the functional conservation of respiratory proteins across different taxonomic groups. This becomes particularly important when examining species-specific adaptations to different environmental conditions or metabolic requirements.

How is recombinant C. mrakii COX2 protein typically expressed and purified?

Recombinant C. mrakii COX2 protein is typically expressed in Escherichia coli expression systems, as indicated by the available commercial product . The expression construct generally includes the mature protein sequence (amino acids 12-247) fused to an N-terminal His-tag to facilitate purification . This approach follows standard recombinant protein expression protocols with some specific considerations:

• Expression system selection: E. coli is preferred due to its rapid growth, high protein yields, and ease of genetic manipulation.

• Vector design: The coding sequence is optimized for E. coli codon usage and inserted into an expression vector with appropriate regulatory elements.

• Purification strategy: The N-terminal His-tag allows for efficient purification using immobilized metal affinity chromatography (IMAC).

• Protein folding considerations: As a membrane protein in its native context, special attention must be paid to protein folding and solubility during recombinant expression.

For researchers working with this protein, purification typically involves cell lysis under native or denaturing conditions, followed by IMAC purification, with optional additional purification steps such as size exclusion chromatography to achieve higher purity if required for specific applications.

What are the structural characteristics of C. mrakii COX2 and how do they compare to other species?

C. mrakii COX2 is a membrane protein component of the cytochrome c oxidase complex. Based on the general structure of COX2 proteins and the information provided in the search results, we can infer several key structural characteristics:

• Size and composition: The mature C. mrakii COX2 protein spans amino acids 12-247 of the full sequence , making it approximately 236 amino acids in length.

• Functional domains: Like other COX2 proteins, it likely contains:

  • A hydrophobic domain anchoring the protein in the membrane

  • A hydrophilic domain extending into the intermembrane space

  • Metal-binding sites, particularly for copper ions that are essential for electron transfer

• Comparative structure: When compared to other yeast species, the COX2 protein is generally highly conserved in its functional domains, but may show variation in less critical regions. This conservation reflects the essential nature of electron transport in cellular respiration .

While specific structural data for C. mrakii COX2 is limited in the provided search results, we can infer from studies of cytochrome c oxidase that it contains specific binding sites for interaction with cytochrome c and other subunits of the COX complex . The specific differences between C. mrakii COX2 and other species would require detailed sequence alignment and structural modeling studies.

What methods are used to assess the functionality of recombinant COX2 proteins?

Assessment of recombinant COX2 functionality employs several complementary methods:

• Spectroscopic analysis: UV-visible spectroscopy, circular dichroism (CD), and electron paramagnetic resonance (EPR) can assess proper folding and metal incorporation. Cytochrome c oxidase components exhibit characteristic spectral properties that can be used to confirm correct assembly and function .

• Electron transfer activity assays: These measure the protein's ability to transfer electrons, typically using reduced cytochrome c as an electron donor and oxygen as the final electron acceptor. The rate of cytochrome c oxidation can be monitored spectrophotometrically.

• Metal content analysis: Techniques such as atomic absorption spectroscopy or inductively coupled plasma mass spectrometry (ICP-MS) can verify the presence of copper ions, which are essential for COX2 function .

• Protein-protein interaction studies: Methods such as co-immunoprecipitation or surface plasmon resonance can assess the protein's ability to interact with other components of the respiratory chain.

• Reconstitution experiments: Incorporating the recombinant protein into liposomes or membrane fragments to assess its ability to function in a membrane environment.

These methods collectively provide a comprehensive evaluation of whether the recombinant protein maintains the structural and functional properties of the native COX2.

How can C. mrakii COX2 be used in evolutionary and phylogenetic studies?

C. mrakii COX2 serves as a valuable marker in evolutionary and phylogenetic studies due to several key characteristics:

• Conserved function with variable sequences: The COX2 gene encodes a protein with highly conserved function but sufficient sequence variation to resolve phylogenetic relationships. This makes it particularly useful for studying the evolutionary history of yeasts and other fungi .

• Methodological approach for phylogenetic analysis:

  • Sequence alignment with orthologs from related species

  • Calculation of substitution rates (dN/dS ratios) to identify regions under selection

  • Construction of phylogenetic trees using maximum likelihood or Bayesian methods

  • Comparative analysis with other mitochondrial and nuclear genes to obtain a comprehensive evolutionary picture

• Identification of horizontal gene transfer (HGT): As observed in other mitochondrial genes, analysis of COX2 sequences can reveal potential instances of HGT between distantly related species, providing insights into the complex evolutionary history of mitochondrial genomes .

• Ploidy and hybridization studies: The analysis of COX2 sequence variation can contribute to understanding ploidy variation and hybridization events in Cyberlindnera species, similar to what has been observed in related yeasts like C. jadinii .

The mitochondrial COX2 gene has been particularly informative in resolving phylogenetic relationships within the Saccharomycotina subphylum, helping to clarify the evolutionary position of Cyberlindnera mrakii relative to other yeast species .

What approaches are used to study COX2 interactions with other respiratory chain components?

Investigating COX2 interactions with other respiratory chain components requires sophisticated biochemical and biophysical techniques:

• Structural biology approaches:

  • X-ray crystallography of the entire cytochrome c oxidase complex

  • Cryo-electron microscopy to visualize the intact respiratory chain supercomplex

  • NMR spectroscopy for studying specific interaction domains

• Biochemical interaction studies:

  • Cross-linking coupled with mass spectrometry to identify interaction interfaces

  • Co-immunoprecipitation with antibodies specific to COX2 or its interaction partners

  • Blue native PAGE to analyze intact respiratory complexes and supercomplexes

• Functional interaction assays:

  • Electron transfer kinetics measurements between cytochrome c and COX

  • Oxygen consumption measurements to assess the functional consequences of mutations at interaction interfaces

  • Reconstitution experiments with purified components to define minimal functional units

• Computational approaches:

  • Molecular docking to predict interaction modes

  • Molecular dynamics simulations to study the dynamics of protein-protein interactions

  • Sequence coevolution analysis to identify co-evolving residues at interaction interfaces

These approaches have revealed that COX2 contains specific binding sites for cytochrome c and interacts extensively with other subunits of the cytochrome c oxidase complex . The copper centers in COX2 play a critical role in these interactions, serving as electron transfer sites within the complex .

How can site-directed mutagenesis of C. mrakii COX2 inform our understanding of its function?

Site-directed mutagenesis represents a powerful approach to dissect the structure-function relationships of C. mrakii COX2:

• Experimental design considerations:

  • Target selection: Focus on conserved residues identified through sequence alignment, particularly those in copper-binding sites and at interaction interfaces with cytochrome c

  • Mutation strategy: Conservative substitutions to probe specific chemical properties versus radical changes to disrupt function

  • Expression system: Ensuring proper folding and assembly of mutant proteins

• Functional assessment protocol:

  • Spectroscopic characterization to evaluate effects on copper binding and electronic structure

  • Electron transfer activity measurements to quantify functional impairment

  • Protein stability and folding analysis to distinguish direct functional effects from structural perturbations

• Specific targets for mutagenesis:

  • Copper coordination sites: Mutations of histidine residues involved in copper binding

  • Cytochrome c docking interface: Modification of charged residues that interact with cytochrome c

  • Transmembrane regions: Alterations to assess membrane integration and complex assembly

• Data interpretation framework:

  • Correlation of structural changes with functional outcomes

  • Comparison with equivalent mutations in well-studied systems like bovine or yeast COX2

  • Integration with computational models to explain experimental observations

This approach has been particularly informative in other cytochrome oxidase systems, where it has helped elucidate the precise mechanisms of electron transfer and the importance of specific residues in maintaining proper function .

What are the challenges in studying the spectroscopic properties of recombinant C. mrakii COX2?

Studying the spectroscopic properties of recombinant C. mrakii COX2 presents several technical challenges:

• Cofactor incorporation issues:

  • Ensuring proper incorporation of copper ions during recombinant expression

  • Maintaining the correct oxidation state of metal centers

  • Verifying the presence of all necessary cofactors in the recombinant protein

• Spectroscopic complexity:

  • Overlapping spectral features from multiple metal centers (copper and heme)

  • Distinguishing signals from COX2 versus other components in the complex

  • Interpreting the complex EPR spectra that arise from copper centers in different environments

• Technical considerations:

  • Sample preparation requirements for different spectroscopic techniques

  • Concentration requirements for optimal signal detection

  • Temperature and buffer conditions affecting spectral properties

• Analytical approach:

  • Complementary use of multiple spectroscopic techniques (UV-visible, EPR, CD, resonance Raman)

  • Comparison with model compounds to aid interpretation

  • Deconvolution of complex spectra to isolate contributions from individual centers

The unique spectroscopic properties of cytochrome c oxidase components, particularly the unusual EPR characteristics of the Cu_A center with g values at 2.18, 2.03, and 1.99 without hyperfine splitting , add to the complexity of these studies but also provide valuable information about the electronic structure and environment of the metal centers.

How does the genomic context of COX2 in C. mrakii compare to other yeast species?

The genomic context of COX2 in C. mrakii, compared to other yeast species, provides insights into mitochondrial genome evolution and organization:

• Comparative genomic analysis:

  • Mitochondrial genome size and organization in C. mrakii compared to related species

  • Synteny analysis of genes surrounding COX2

  • GC content and codon usage patterns specific to C. mrakii

• Intron presence and distribution:

  • C. mrakii COX2 may contain introns, similar to what has been observed in other mitochondrial genes across yeast species

  • Analysis of intron conservation and potential horizontal gene transfer events

  • Functional significance of introns in mitochondrial gene expression

• Regulatory elements:

  • Promoter structures and transcription initiation sites

  • RNA processing signals and transcript stability elements

  • Translation initiation and termination signals

• Species-specific features:

  • Genome assembly shows that the mitochondrial genome diversity across Saccharomycotina has been significantly expanded, increasing from 132 to 353 species with available mtDNAs

  • Most introns observed do not share high sequence similarity between species (65.6%), with about 30% shared within taxonomic groups

  • Small number of introns are shared across groups, particularly in the order Saccharomycetales

This comparative genomic context is essential for understanding the evolutionary forces acting on COX2 and can provide insights into the functional constraints and adaptive changes in mitochondrial genes across different yeast lineages.

What are the optimal expression conditions for producing functional recombinant C. mrakii COX2?

Optimizing expression conditions for functional recombinant C. mrakii COX2 requires careful consideration of multiple factors:

• Expression system selection:

  • E. coli is commonly used for initial expression trials

  • Yeast expression systems (Pichia pastoris or Saccharomyces cerevisiae) may provide a more native-like environment for proper folding

  • Insect cell or mammalian cell systems for complex cases requiring extensive post-translational modifications

• Expression construct design:

  • Vector selection with appropriate promoter strength

  • Inclusion of a purification tag (typically His-tag)

  • Signal sequence optimization for membrane targeting

  • Codon optimization for the chosen expression host

• Culture conditions optimization:

  Parameter	Range to Test	Considerations
  Temperature	16-30°C	Lower temperatures often favor proper folding
  Induction timing	OD600 0.6-1.2	Cell density affects protein expression efficiency
  Inducer concentration	0.1-1.0 mM IPTG	Lower concentrations may improve folding
  Media composition	LB, TB, M9	Richer media can increase yield but may affect folding
  Aeration	150-250 rpm	Oxygen availability affects cell metabolism
  Expression time	4-24 hours	Longer times increase yield but may lead to degradation

• Additives and supplements:

  • Metal ions (particularly copper) to facilitate cofactor incorporation

  • Osmolytes or chaperone co-expression to improve folding

  • Membrane-mimetic environments for proper folding of transmembrane domains

• Extraction and solubilization:

  • Gentle lysis methods to preserve protein structure

  • Appropriate detergent selection for membrane protein solubilization

  • Buffer optimization to maintain protein stability during purification

These optimized conditions ensure the production of properly folded, functional recombinant COX2 protein suitable for downstream structural and functional studies.

How can researchers effectively analyze the interaction between C. mrakii COX2 and cytochrome c?

Analyzing the interaction between C. mrakii COX2 and cytochrome c requires a multi-faceted approach:

• Binding affinity determination:

  • Surface plasmon resonance (SPR) to measure real-time binding kinetics

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

  • Microscale thermophoresis (MST) for solution-based interaction analysis

  • Fluorescence anisotropy if one protein can be fluorescently labeled

• Structural characterization of the complex:

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

  • Hydrogen-deuterium exchange mass spectrometry to map binding regions

  • FRET-based approaches to measure distances between specific residues

  • Co-crystallization attempts or cryo-EM analysis of the complex

• Functional analysis of the interaction:

  • Electron transfer kinetics measurements using stopped-flow spectroscopy

  • Oxygen consumption assays to assess the functional consequence of the interaction

  • Site-directed mutagenesis of predicted interface residues to validate their importance

• Computational approaches:

  • Molecular docking to predict binding modes

  • Molecular dynamics simulations to study the dynamics of the complex

  • Electrostatic surface mapping to identify complementary interaction regions

• Comparative analysis:

  • Comparison with known cytochrome c-COX2 interactions from other species

  • Evaluation of species-specific adaptations in the interaction interface

  • Assessment of the evolutionary conservation of key interaction residues

These methodologies collectively provide a comprehensive characterization of the interaction, yielding insights into both the structural basis and functional significance of the C. mrakii COX2-cytochrome c complex.

What approaches can be used to study the copper binding sites in C. mrakii COX2?

The copper binding sites in C. mrakii COX2 can be studied using a combination of spectroscopic, biochemical, and computational approaches:

• Spectroscopic characterization:

  • Electronic absorption spectroscopy to identify characteristic copper center transitions

  • EPR spectroscopy to characterize the electronic structure of copper centers

  • X-ray absorption spectroscopy (XAS) including XANES and EXAFS to determine coordination geometry and ligand identity

  • Resonance Raman spectroscopy to probe metal-ligand vibrations

• Metal content analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS) for precise quantification

  • Atomic absorption spectroscopy as an alternative quantification method

  • Colorimetric assays for rapid screening of copper content

• Site-directed mutagenesis strategy:

  • Systematic mutation of predicted copper-coordinating residues (typically histidines)

  • Conservative substitutions (His→Gln) to maintain hydrogen bonding capability

  • Non-conservative substitutions (His→Ala) to completely remove coordination

  • Analysis of the impact on copper binding and enzymatic activity

• Structural biology approaches:

  • X-ray crystallography of the protein or relevant fragments

  • NMR spectroscopy of isolated domains containing copper sites

  • Homology modeling based on known structures of COX2 from other species

• Bioinformatic analysis:

  • Sequence alignment to identify conserved copper-binding motifs

  • Structural prediction to locate potential copper coordination sites

  • Evolutionary analysis to track conservation of copper-binding residues

Based on studies of cytochrome c oxidase from other species, the Cu_A center in COX2 likely has unusual spectroscopic characteristics with g values at approximately 2.18, 2.03, and 1.99, reminiscent of an organic free radical rather than a typical Cu^II center . This distinctive spectroscopic signature can be used to confirm proper copper incorporation and coordination in the recombinant protein.

How does C. mrakii COX2 compare to COX2 proteins from other yeasts in the Cyberlindnera genus?

Comparative analysis of C. mrakii COX2 with other Cyberlindnera species reveals important evolutionary and functional insights:

This comparative analysis contributes to our understanding of how evolutionary processes have shaped the structure and function of COX2 across the Cyberlindnera genus, providing insights into both conserved mechanisms and species-specific adaptations.

What insights can be gained from studying COX2 evolution across diverse yeast species?

Studying COX2 evolution across diverse yeast species provides valuable insights into mitochondrial genome evolution and adaptation:

These evolutionary insights contribute to our broader understanding of mitochondrial genome evolution, the interplay between mitochondrial and nuclear genomes, and the genetic basis of speciation.

How can structural information from related COX2 proteins inform research on C. mrakii COX2?

Structural information from related COX2 proteins provides a valuable framework for research on C. mrakii COX2:

• Homology modeling approach:

  • Selection of appropriate template structures (typically from well-characterized species)

  • Sequence alignment to identify conserved structural elements

  • Model building with attention to copper-binding sites and interaction interfaces

  • Validation using energy minimization and stereochemical quality assessment

• Structural conservation analysis:

  • Identification of conserved structural motifs across diverse species

  • Mapping of sequence conservation onto structural models

  • Analysis of structurally constrained regions versus flexible/variable regions

• Functional site identification:

  • Based on structural information from other species, cytochrome c oxidase contains two heme centers (a and a₃) and two copper centers (Cu_A and Cu_B)

  • The Cu_A center, located in COX2, functions primarily in electron transfer

  • Structural data suggests that Cu_A likely involves coordination by cysteine residues, forming a unique electronic structure that resembles an organic free radical

• Structure-guided experimental design:

  • Rational design of site-directed mutagenesis experiments

  • Selection of optimal protein fragments for expression and characterization

  • Design of protein engineering strategies to enhance stability or function

• Structural basis for species-specific differences:

  • Identification of structural variations that may underlie functional differences

  • Analysis of surface properties that may affect interactions with partner proteins

  • Investigation of structural elements that may contribute to adaptation to different environments

This structure-informed approach accelerates research by providing testable hypotheses about the structure-function relationships in C. mrakii COX2, guiding experimental design, and facilitating the interpretation of experimental results.

What are the remaining knowledge gaps in our understanding of C. mrakii COX2?

Despite advances in our understanding of C. mrakii COX2, several significant knowledge gaps remain:

• Structural characterization:

  • Lack of high-resolution structural data specific to C. mrakii COX2

  • Incomplete understanding of species-specific structural features

  • Limited information on conformational dynamics during electron transfer

• Functional specialization:

  • Unclear whether C. mrakii COX2 possesses unique functional properties compared to other species

  • Limited knowledge of kinetic parameters specific to this protein

  • Incomplete understanding of how sequence variations translate to functional differences

• Regulatory mechanisms:

  • Limited information on transcriptional and post-translational regulation

  • Poor understanding of assembly pathways for the complete cytochrome c oxidase complex

  • Unknown mechanisms for coordinating expression with nuclear-encoded partners

• Ecological and evolutionary context:

  • Incomplete picture of how COX2 variations contribute to ecological adaptation

  • Limited understanding of selective pressures specific to the Cyberlindnera lineage

  • Unclear relationship between COX2 evolution and speciation in this genus

• Technical limitations:

  • Challenges in expressing and purifying sufficient quantities of functional protein

  • Difficulties in reconstituting the complete cytochrome c oxidase complex

  • Limited tools for manipulating the mitochondrial genome in Cyberlindnera species

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, biochemistry, molecular genetics, and evolutionary analysis to build a comprehensive understanding of C. mrakii COX2.

What are promising future research directions for C. mrakii COX2?

Several promising research directions could significantly advance our understanding of C. mrakii COX2:

• Structural biology initiatives:

  • Determination of high-resolution structure using cryo-EM or X-ray crystallography

  • Time-resolved structural studies to capture different functional states

  • Structural characterization of COX2 in the context of the complete cytochrome c oxidase complex

• Functional genomics approaches:

  • Development of mitochondrial genome editing tools for Cyberlindnera species

  • Creation of site-directed mutants to probe structure-function relationships

  • Complementation studies in heterologous systems to assess functional conservation

• Systems biology integration:

  • Multi-omics approaches to understand COX2 in the broader context of cellular metabolism

  • Network analysis to map interactions between mitochondrial and nuclear genetic systems

  • Modeling of electron transport dynamics incorporating species-specific parameters

• Comparative and evolutionary studies:

  • Expanded phylogenetic analysis with increased taxon sampling

  • Molecular evolution studies to identify sites under selection

  • Experimental evolution approaches to study adaptation under controlled conditions

• Biotechnological applications:

  • Engineering COX2 for enhanced stability or activity

  • Development of biosensors based on electron transfer properties

  • Exploration of potential applications in bioenergetics or bioelectronics

These research directions would not only advance our fundamental understanding of C. mrakii COX2 but could also contribute to broader fields including mitochondrial biology, protein evolution, and biotechnology applications of electron transfer proteins.