Recombinant Saccharomyces cerevisiae NADH-cytochrome b5 reductase 2 (MCR1)

What is the primary function of S. cerevisiae NADH-cytochrome b5 reductase 2 (MCR1) and how does it differ from CBR1?

MCR1 functions as a mitochondrial NADH-cytochrome b5 reductase in S. cerevisiae, catalyzing electron transfer from NADH to cytochrome b5. While both MCR1 and CBR1 are NADH-cytochrome b5 reductases in yeast, they differ in cellular localization and specific functions. CBR1 is primarily microsomal (ER-associated) and demonstrates strict specificity for NADH over NADPH as an electron donor, with a characteristic FAD-binding domain for electron transfer . MCR1, by contrast, is predominantly located in the mitochondrial outer membrane and intermembrane space, where it participates in mitochondrial electron transport chains.

Like other NADH-cytochrome b5 reductases, MCR1 contains conserved FAD-binding and NADH-binding domains with specific arrangements of three key amino acid residues (arginine, tyrosine, and serine) that facilitate flavin binding through hydrogen bonds . The enzyme preferentially utilizes NADH over NADPH as its electron donor, similar to what has been observed with other NADH-cytochrome b5 reductases .

What expression systems are most effective for producing recombinant S. cerevisiae MCR1?

Several expression systems have proven effective for producing recombinant NADH-cytochrome b5 reductases, which can be adapted for MCR1 expression:

For functional studies requiring proper folding and native-like activity, expression in filamentous fungi has demonstrated significant success, with a 4.7-fold increase in ferricyanide reduction activity observed when expressing a related cytochrome b5 reductase . For structural studies requiring larger protein quantities, bacterial expression with subsequent refolding protocols may be more appropriate.

What purification strategies yield the highest purity and activity for recombinant MCR1?

Based on successful purification strategies for related NADH-cytochrome b5 reductases, a multi-step approach yields optimal results:

• Microsomal fraction isolation: Differential centrifugation to isolate membrane fractions containing the MCR1 protein

• Solubilization: Using cholic acid sodium salt or similar detergents to solubilize the membrane-bound enzyme

• Sequential chromatography:

  • DEAE-Sephacel ion exchange chromatography

  • Mono-Q HR 5/5 chromatography for higher resolution

  • AMP-Sepharose 4B affinity chromatography exploiting the enzyme's affinity for the adenine moiety

This purification strategy has achieved a 645-fold increase in NADH-ferricyanide reductase specific activity for related cytochrome b5 reductases . Throughout purification, activity assays should be performed using NADH as the electron donor and ferricyanide or recombinant cytochrome b5 as the electron acceptor.

How can researchers accurately assess the electron transfer kinetics of recombinant MCR1?

To accurately characterize the electron transfer kinetics of recombinant MCR1, researchers should implement a comprehensive approach:

• Spectrophotometric assays: Monitor the reduction of cytochrome b5 by following absorbance changes at 424 nm (reduced form) versus 409 nm (oxidized form) . This allows calculation of reaction rates under varying substrate concentrations.

• Determination of substrate specificity: Compare electron transfer rates using both NADH and NADPH as potential electron donors, with strict controls. Based on studies of related enzymes, MCR1 is expected to display strict specificity for NADH, with a typical Km value of approximately 1.5 μM for NADH .

• Reconstitution experiments: Combine purified recombinant MCR1 with its electron acceptors (cytochrome b5) in vitro to assess native-like activity. This approach allows researchers to determine:

  • Km values for both NADH and electron acceptors

  • Catalytic efficiency (kcat/Km)

  • Effects of pH, temperature, and ionic strength on activity

• Inhibition studies: Evaluate the impact of known inhibitors of electron transport chains to further characterize the enzyme's active site and electron transfer mechanism.

A typical experimental setup would include:

• Purified recombinant MCR1 (5-20 nM)

• Varying concentrations of NADH (0.1-100 μM)

• Recombinant cytochrome b5 or other electron acceptors (5-50 μM)

• Appropriate buffer system (typically 50 mM phosphate buffer, pH 7.0-7.4)

• Spectrophotometric measurements at 340 nm (NADH oxidation) and 424 nm (cytochrome b5 reduction)

What structural features determine the FAD binding and catalytic activity of S. cerevisiae MCR1?

The FAD binding and catalytic activity of S. cerevisiae MCR1 are determined by several key structural features that can be inferred from related NADH-cytochrome b5 reductases:

• Conserved flavin-binding β-barrel domain: This domain contains specific arrangements of amino acid residues that create the binding pocket for the FAD cofactor. Crystal structure analysis of the related CBR1 reveals the specific binding interactions .

• Critical amino acid triad: A specific arrangement of three highly conserved amino acid residues—arginine, tyrosine, and serine—plays a crucial role in binding flavin through hydrogen bonds . These residues are typically found in the FAD-binding domain and are essential for proper cofactor orientation.

• NADH-binding domain: This domain contains specific residues that interact with the nicotinamide portion of NADH and position it optimally for hydride transfer to FAD.

• Domain interface residues: The residues at the interface between the FAD-binding and NADH-binding domains are critical for facilitating electron transfer and maintaining proper enzyme conformation.

Based on crystal structure data from S. cerevisiae CBR1 (PDB ID: 7ROM) , researchers can identify these key structural elements in MCR1 through homology modeling. X-ray crystallography remains the gold standard for definitive structural characterization.

How do post-translational modifications affect the activity and localization of recombinant MCR1?

Post-translational modifications (PTMs) significantly influence both the activity and subcellular localization of recombinant MCR1:

• N-terminal processing: MCR1 contains an N-terminal hydrophobic domain that serves as a membrane anchor. Proper processing of this domain is critical for correct subcellular localization. Expression systems lacking appropriate processing machinery may yield improperly processed, mislocalized enzyme .

• Disulfide bond formation: Correct disulfide bond formation is essential for proper folding and stability. Research on related reductases suggests that oxidative folding environments enhance proper disulfide bond formation.

• Glycosylation: While not extensively characterized for MCR1 specifically, potential N-glycosylation sites may affect protein stability and solubility when expressed in different systems.

To assess the impact of these modifications, researchers should compare:

• Expression in prokaryotic systems (lacking most PTMs) versus eukaryotic systems

• Wild-type versus mutated forms with altered modification sites

• Activity and localization patterns using subcellular fractionation followed by Western blotting and activity assays

For recombinant expression, selecting a system capable of performing the relevant PTMs is critical for obtaining functionally active MCR1. S. cerevisiae or Pichia pastoris expression systems may provide advantages for producing properly modified enzyme.

What is the role of MCR1 in mitochondrial electron transport and redox homeostasis?

MCR1 plays several key roles in mitochondrial electron transport and redox homeostasis:

• Electron shuttle: MCR1 transfers electrons from NADH to cytochrome b5, contributing to mitochondrial electron transport pathways distinct from the classical respiratory chain.

• Redox balance maintenance: By oxidizing NADH to NAD+, MCR1 helps maintain the redox balance in the intermembrane space of mitochondria.

• Interaction with other mitochondrial proteins: MCR1 likely interacts with specific mitochondrial proteins to facilitate electron transfer processes, though these interactions require further characterization.

Research approaches to study MCR1's role include:

• Deletion mutant analysis (Δmcr1) to assess phenotypic changes

• Metabolomic analysis to identify altered metabolite profiles

• Redox proteomics to detect changes in protein oxidation states

• In vivo electron transfer studies using fluorescent redox sensors

These phenotypic differences highlight MCR1's contribution to mitochondrial redox homeostasis, particularly under stress conditions.

How can researchers differentiate between MCR1 and CBR1 activity in mixed microsomal preparations?

Differentiating between MCR1 and CBR1 activities in mixed microsomal preparations requires selective experimental approaches:

• Subcellular fractionation: The primary approach involves careful separation of mitochondrial fractions (containing MCR1) from microsomal/ER fractions (containing CBR1) through differential centrifugation.

• Inhibitor profiling: Researchers can develop specific inhibitors or use existing compounds that differentially affect MCR1 versus CBR1. The inhibition profiles can serve as a distinguishing feature.

• Immunochemical methods: Using antibodies specific to either MCR1 or CBR1, researchers can perform:

  • Immunodepletion studies to selectively remove one enzyme

  • Western blotting to quantify relative amounts

  • Immunoprecipitation to isolate specific enzyme activities

• Genetic manipulation: In research using S. cerevisiae:

  • Single knockout strains (Δmcr1 or Δcbr1)

  • Double knockout with selective complementation

  • Epitope-tagged versions of each enzyme

• Kinetic differentiation: While both enzymes typically prefer NADH over NADPH , subtle differences in:

  • Km values for NADH

  • Substrate specificities for electron acceptors

  • pH optima

  • Temperature sensitivity

These approaches, when combined, provide a robust strategy for distinguishing between the activities of these related but distinct reductases in complex biological samples.

What are the optimal conditions for assessing MCR1 activity in vitro?

The optimal conditions for assessing MCR1 activity in vitro have been established through rigorous experimentation:

For recombinant MCR1, it is essential to confirm that the protein contains stoichiometric amounts of FAD cofactor, as substoichiometric FAD binding can dramatically reduce specific activity. Researchers should perform absorption spectroscopy to verify the characteristic flavin peaks (375 and 450 nm) before activity measurements .

What common challenges arise when expressing recombinant MCR1 and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant MCR1:

• Low expression yield:

  • Problem: MCR1 contains a hydrophobic N-terminal domain that can cause aggregation.

  • Solution: Express a truncated form (residues 28-284, similar to the successful approach with CBR1 ) or use solubility-enhancing fusion tags (MBP, SUMO).

• Improper cofactor incorporation:

  • Problem: Insufficient FAD incorporation leads to lower specific activity.

  • Solution: Supplement growth media with riboflavin (10-50 mg/L) or add FAD during purification and allow reconstitution.

• Protein misfolding:

  • Problem: Formation of inclusion bodies in bacterial expression systems.

  • Solution: Lower induction temperature (16-18°C), reduce inducer concentration, or switch to a eukaryotic expression system such as S. cerevisiae or Pichia pastoris.

• Membrane association challenges:

  • Problem: Difficulty extracting active enzyme from membranes.

  • Solution: Use mild detergents like cholic acid sodium salt for solubilization, following the successful approach used for related reductases .

• Loss of activity during purification:

  • Problem: FAD dissociation during purification steps.

  • Solution: Include low concentrations of FAD (1-5 μM) in all purification buffers.

Case studies have shown that expressing the soluble domain alone (without the N-terminal membrane anchor) can increase soluble protein yield without compromising the fundamental catalytic properties, as demonstrated with the CBR1 fragment (residues 28-284) .

How can researchers accurately compare electron transfer efficiency between wild-type and mutant forms of MCR1?

To accurately compare electron transfer efficiency between wild-type and mutant forms of MCR1, researchers should implement a systematic approach:

A typical experimental design would include measuring initial reaction rates across a range of substrate concentrations (0.1-10× Km) for both wild-type and mutant enzymes under identical conditions. The resulting kinetic parameters provide quantitative metrics for comparing electron transfer efficiency.

What spectroscopic methods are most informative for studying the redox properties of recombinant MCR1?

Multiple spectroscopic techniques provide complementary insights into the redox properties of recombinant MCR1:

• UV-Visible Absorption Spectroscopy:

  • Monitors characteristic FAD spectral features (peaks at 375 and 450 nm)

  • Tracks the reduction of FAD during catalysis

  • Enables real-time observation of cytochrome b5 reduction (shift from 409 to 424 nm)

  • Allows determination of the FAD:protein stoichiometry

• Fluorescence Spectroscopy:

  • Exploits the natural fluorescence of the FAD cofactor

  • Can detect subtle changes in the FAD microenvironment

  • Fluorescence quenching studies reveal accessibility of the flavin

  • Useful for protein-protein interaction studies with electron acceptors

• Circular Dichroism (CD) Spectroscopy:

  • Provides information about protein secondary structure

  • Detects conformational changes upon substrate binding

  • Near-UV CD gives insight into the environment of aromatic residues

  • Visible-range CD provides information about the FAD binding environment

• Electron Paramagnetic Resonance (EPR):

  • Detects paramagnetic species including flavin semiquinone

  • Characterizes the electronic structure of redox intermediates

  • Can be combined with rapid freeze-quench techniques to capture transient species

  • Especially valuable for studying one-electron transfer steps

• Resonance Raman Spectroscopy:

  • Provides detailed information about the FAD vibrational modes

  • Sensitive to changes in flavin environment during redox cycling

  • Can detect subtle structural changes in the active site

The combination of these techniques provides a comprehensive understanding of the redox cycle, allowing researchers to identify key mechanistic features and compare wild-type MCR1 with mutant forms or related enzymes like CBR1.

How can structural biology approaches be used to improve our understanding of MCR1 function?

Structural biology approaches offer powerful insights into MCR1 function through multiple complementary techniques:

The crystal structure of S. cerevisiae CBR1 (residues 28-284) bound to FAD provides an excellent template for homology modeling of MCR1, allowing researchers to identify conserved structural features and predict the impacts of mutations on function.

What are the most effective strategies for analyzing MCR1-protein interactions in a cellular context?

Understanding MCR1's interactions with other proteins requires sophisticated cellular and biochemical approaches:

• Proximity-dependent labeling techniques:

  • BioID: Fusion of MCR1 with a promiscuous biotin ligase to identify proximal proteins

  • APEX2: Peroxidase-based proximity labeling followed by mass spectrometry

  • These approaches can map the protein interaction landscape of MCR1 in its native mitochondrial environment

• Co-immunoprecipitation coupled with mass spectrometry:

  • Enables identification of stable interaction partners

  • Can be performed with epitope-tagged MCR1 expressed at endogenous levels

  • Crosslinking prior to lysis can capture transient interactions

• Förster Resonance Energy Transfer (FRET):

  • Live-cell imaging of protein-protein interactions

  • Can be implemented using fluorescent protein fusions

  • Allows real-time monitoring of dynamic interactions

  • Particularly useful for studying MCR1's interaction with cytochrome b5

• Split-protein complementation assays:

  • BiFC (Bimolecular Fluorescence Complementation)

  • Split-luciferase assays for quantitative measurement

  • Yeast two-hybrid or split-ubiquitin systems

• In situ approaches:

  • Proximity Ligation Assay (PLA) to visualize protein interactions in fixed cells

  • Immunofluorescence co-localization studies

  • Super-resolution microscopy for detailed spatial mapping

• Functional validation:

  • Mutational analysis of putative interaction interfaces

  • Competitive peptide inhibition of specific interactions

  • Correlation of interaction strength with functional readouts

These approaches, when combined, provide a comprehensive view of MCR1's protein interaction network and help elucidate how these interactions contribute to mitochondrial electron transport and redox homeostasis.

How can recombinant MCR1 be utilized in synthetic biology applications?

Recombinant MCR1 offers several promising applications in synthetic biology:

• Engineered electron transfer systems:

  • Incorporation into artificial electron transport chains

  • Creation of NADH-dependent biocatalytic cascades

  • Development of bio-electrochemical systems using MCR1 as an electron conduit

• Metabolic engineering applications:

  • Modulation of NAD+/NADH ratios in engineered pathways

  • Enhancement of mitochondrial electron transport in production strains

  • Integration into pathways requiring controlled electron transfer

• Biosensor development:

  • NADH-sensing elements using MCR1 coupled to reporter systems

  • Detection of compounds that affect mitochondrial electron transport

  • Screening platforms for compounds that modulate reductase activity

• Protein engineering opportunities:

  • Creation of chimeric reductases with novel electron acceptor specificities

  • Development of MCR1 variants with altered cofactor preferences

  • Engineering enhanced stability for industrial applications

The strict substrate specificity of MCR1 for NADH over NADPH makes it particularly valuable for applications requiring selective utilization of NADH in the presence of both pyridine nucleotides. Additionally, its natural role in mitochondrial electron transport provides a foundation for engineering artificial electron transport systems.

What insights from MCR1 research can be applied to understanding related human enzymes?

Research on S. cerevisiae MCR1 provides valuable insights applicable to human NADH-cytochrome b5 reductases:

• Structural conservation and divergence:

  • The high sequence similarity (approximately 35%) between fungal and mammalian NADH-cytochrome b5 reductases indicates conserved catalytic mechanisms

  • Differences in membrane-binding domains may explain distinct subcellular localizations

  • Comparative structural analysis can identify critical residues for targeted studies in human enzymes

• Disease-relevant mutations:

  • Mutations in human NADH-cytochrome b5 reductases are associated with methemoglobinemia

  • Yeast MCR1 can serve as a model system for studying equivalent mutations

  • Structure-function relationships elucidated in MCR1 can inform predictions about human disease mutations

• Drug discovery applications:

  • MCR1 can serve as a screening platform for compounds that modulate reductase activity

  • Inhibitors developed against MCR1 may provide lead compounds for human enzyme targets

  • Differences in binding sites between fungal and human enzymes can be exploited for antifungal development

• Redox biology insights:

  • Mechanisms of electron transfer elucidated in MCR1 likely apply to human enzymes

  • Mitochondrial redox regulation principles are often conserved from yeast to humans

  • Stress response pathways involving these reductases may share common features

By leveraging the experimental accessibility of the yeast system, researchers can gain mechanistic insights that would be challenging to obtain directly with human enzymes, advancing our understanding of these important components of cellular redox biology.