Recombinant Cupriavidus necator Cytochrome c-type biogenesis protein CcmE (ccmE), partial

What is the function of CcmE in cytochrome c biogenesis in Cupriavidus necator?

CcmE functions as a heme chaperone in the System I cytochrome c maturation pathway in C. necator. It covalently binds heme through a conserved residue and transports it to the cytochrome c synthetase complex. In the C. necator System I pathway, CcmE works within the CcmABCEF complex to facilitate heme delivery and attachment to apocytochrome c, which is essential for cellular respiration and electron transport chain function .

Unlike in E. coli where heme binding occurs through a conserved histidine residue (H130) in the HXXXY motif, C. necator and other related organisms contain a cysteine residue in this conserved region. This cysteine-based heme binding represents a significant variation in the cytochrome c biogenesis pathway across bacterial species .

How does the CcmE protein contribute to the energy metabolism of C. necator?

CcmE plays a critical role in the broader energy metabolism of C. necator by enabling the maturation of functional cytochrome c proteins. These cytochromes are essential components of the electron transport chain, particularly the complex respiratory system that C. necator employs across different growth conditions.

In C. necator, the electron transport chain is remarkably complex, with multiple terminal cytochrome oxidases (including bb3, cbb3, and aa3 complexes) that require proper cytochrome c biogenesis for function. Fitness studies have shown that these terminal oxidases have different importance depending on the growth substrate:

This differential utilization pattern demonstrates how cytochrome c-based respiration adapts to different energy sources, highlighting the importance of functional CcmE for versatile energy metabolism .

What are the best methods for recombinant expression and purification of C. necator CcmE?

For optimal recombinant expression of C. necator CcmE, the following methodological approach is recommended:

• Expression System Selection:

  • E. coli is the preferred heterologous host for expression due to compatibility with the System I cytochrome c biogenesis pathway .

  • For enhanced expression, use E. coli strains with complete CcmABCDEFGH system (such as E. coli BL21(DE3) harboring pEC86).

• Vector Design:

  • Clone the ccmE gene (with or without signal sequence depending on experimental goals) into an expression vector with an inducible promoter (T7 or tetracycline-inducible systems work well).

  • Include appropriate affinity tags (6xHis or Strep tags) at either the N-terminus (if signal sequence is removed) or C-terminus.

• Culture Conditions:

  • Grow cultures in fructose-based minimal media at 30°C (optimal for C. necator proteins).

  • Induce at mid-log phase (OD600 ~0.6) with appropriate inducer.

  • Add 5-aminolevulinic acid (50-100 μM) to enhance heme biosynthesis.

  • Extended expression at lower temperatures (16-20°C) improves soluble protein yield.

• Purification Protocol:

  • Extract membrane fractions using differential centrifugation after cell lysis.

  • Solubilize membrane proteins with mild detergents (0.5-1% Triton X-100 or n-dodecyl-β-D-maltopyranoside).

  • Purify using affinity chromatography (Ni-NTA for His-tagged variants) followed by gel filtration.

  • To maintain protein stability, use buffers containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 5-10% glycerol.

• Quality Assessment:

  • Verify heme binding by UV-visible spectroscopy (characteristic peaks at ~410 nm for holo-CcmE).

  • Confirm purity by SDS-PAGE and heme staining .

This protocol typically yields 2-5 mg of purified CcmE per liter of culture with >85% purity .

How can knockout studies of ccmE be designed to understand its role in C. necator?

Designing effective ccmE knockout studies in C. necator requires careful consideration of both the genetic approach and phenotypic analysis:

• Genetic Approach Options:

  • Suicide Plasmid Method: Use pLO3-based suicide vectors for gene deletion through homologous recombination. This approach requires removal of restriction sites (particularly GAYNNNNNCTTGY) from the vector and using unmethylated DNA (from E. coli dam-/dcm- strains) to bypass C. necator's restriction systems .

  • CRISPR-Cas9 System: While more challenging in C. necator due to transformation efficiency, this can provide cleaner knockouts with fewer polar effects.

  • Transposon Mutagenesis: Create a barcoded transposon library for high-throughput screening of ccmE and related genes, enabling fitness studies across various conditions .

• Verification of Knockouts:

  • PCR verification of deletion using primers flanking the deletion site.

  • Sequencing of the genomic region to confirm clean deletion.

  • Western blot analysis to confirm absence of CcmE protein.

  • Heme staining to verify loss of holocytochrome c production .

• Phenotypic Analysis Strategy:

  • Growth Assessment: Compare growth rates across different substrates (formate, fructose, succinate, H2/CO2) to assess condition-dependent effects.

  • Respiratory Capacity: Measure oxygen consumption rates to quantify respiratory defects.

  • Cytochrome c Production: Use heme staining of cell lysates to detect presence/absence of mature cytochromes.

  • Complementation Studies: Re-introduce wild-type or mutant ccmE genes to confirm phenotype rescue.

• Advanced Phenotypic Analysis:

  • Protein Allocation Analysis: Use LC-MS/MS proteomics to assess changes in protein allocation following ccmE deletion.

  • Resource Balance Analysis (RBA): Apply modeling approaches to understand metabolic rearrangements in response to cytochrome c deficiency .

Knockout studies should particularly focus on different growth conditions that utilize various components of the electron transport chain to fully understand the condition-specific importance of CcmE .

How does CcmE interact with other components of the System I cytochrome c biogenesis pathway in C. necator?

The interaction between CcmE and other components of the System I pathway in C. necator involves specific protein-protein contacts that facilitate heme transfer:

• CcmC-CcmE Interaction:

  • In C. necator, similar to other bacteria, CcmE interacts with CcmC to receive heme.

  • Key interaction residues have been identified through mutational studies:

    • On CcmC: Asp47, Gln50, and Arg55 are critical for CcmE interaction

    • On CcmE: Arg73, Asp101, and Glu105 form the complementary interaction interface

  • These interactions occur primarily through protein-protein contacts rather than protein-heme interactions as confirmed by NMR spectroscopy .

  • Triple mutations in these residues (R73A/D101A/E105A) completely abolish holo-CcmE formation.

• CcmABC-CcmE Complex Formation:

  • Unlike E. coli, C. necator lacks CcmD, which normally facilitates the release of holo-CcmE from the CcmABCE adduct.

  • The CcmABC complex in C. necator must operate through an alternative mechanism for holo-CcmE formation and release.

  • The ATP-binding cassette (ABC) transporter formed by CcmA (ATP hydrolysis) and CcmB is essential for this process .

• CcmF-CcmE Interaction:

  • CcmE transfers heme to apocytochrome c through interaction with CcmF.

  • C. necator contains multiple CcmF homologs with differential importance:

    • CcmF2 is essential for cytochrome c maturation

    • CcmF1 contributes to but is not essential for this process

  • The interaction involves the transfer of the covalently bound heme from CcmE to the cytochrome c substrate .

A significant difference in C. necator is the absence of several components found in other bacterial systems (CcmD, CcmH, CcmI), suggesting a streamlined or modified System I pathway that merits further investigation .

How does the heme binding mechanism of CcmE in C. necator differ from other bacteria?

The heme binding mechanism in C. necator CcmE represents a significant variation from the canonical System I pathway found in model organisms like E. coli:

• Heme-Binding Residue Difference:

  • In E. coli and most Gram-negative bacteria, a conserved histidine residue (H130) in the HXXXY motif of CcmE covalently binds heme.

  • In contrast, C. necator and related organisms have replaced this histidine with a cysteine residue in a CXXXY motif .

  • This cysteine-based heme binding represents a distinct biochemical mechanism.

• Functional Implications:

  • The cysteine-heme bond likely has different chemical properties (thioether vs. histidine-heme bond).

  • Substitution experiments demonstrated that C120A mutations abolish heme binding, confirming the essential role of this cysteine residue.

  • Despite the difference in binding residue, the functional outcome (heme delivery to cytochrome c) is preserved.

• Evolutionary Perspective:

  • This cysteine variant is shared with several bacterial species (such as Desulfovibrio desulfuricans) and many archaea.

  • The distribution suggests multiple independent evolutionary events leading to this alternative heme binding mechanism.

  • Phylogenetic analyses indicate horizontal gene transfer events between different bacterial groups may have contributed to this distribution .

• Structural Consequences:

  • NMR studies suggest that the cysteine-heme binding creates a distinct conformation compared to the histidine-heme binding.

  • These structural differences may influence the kinetics and thermodynamics of heme transfer to cytochrome c.

This alternative heme binding mechanism may represent an adaptation to the specific energy metabolism requirements of C. necator in its diverse growth environments .

What is the significance of CcmE in the versatile energy metabolism of C. necator across different trophic modes?

CcmE plays a crucial role in supporting C. necator's metabolic versatility across different trophic modes through its function in cytochrome c biogenesis:

• Substrate-Dependent Importance:

  • Fitness studies using transposon-mutant libraries have revealed differential importance of cytochrome c-based respiration depending on the growth substrate.

  • The following table summarizes the relative importance of various respiratory complexes across trophic modes:

• Respiratory Adaptations:

  • For formatotrophic growth, the cbb3 complex (requiring cytochrome c) shows the strongest fitness effect, suggesting a high-efficiency pathway via cytochrome reductase and oxidase.

  • In heterotrophic growth (fructose), the direct oxidation pathway through quinol oxidases appears more prominent.

  • These adaptations allow C. necator to optimize energy yield from diverse substrates .

• Redox Balance Maintenance:

  • Functional cytochromes c are essential for maintaining redox balance, especially when using substrates with different reduction potentials.

  • The adaptable respiratory chain containing various cytochromes c provides flexibility in electron flow and proton pumping efficiency .

• Protein Resource Allocation:

  • Proteomics studies indicate that C. necator allocates significant resources to respiratory components even when they're not fully utilized.

  • This constitutive expression represents an investment in metabolic readiness despite the protein cost.

  • Up to 43% of the proteome may be non-utilized in specific growth conditions, some being respiratory components .

This data demonstrates how CcmE supports C. necator's remarkable metabolic flexibility, allowing it to thrive in environments with varying energy sources and contributing to its potential as a biotechnological chassis organism .

What are the methodological challenges in analyzing CcmE-heme interactions in C. necator?

Analyzing CcmE-heme interactions in C. necator presents several technical challenges that require specialized methodological approaches:

• Membrane Protein Solubilization:

  • CcmE is partially membrane-associated, making isolation in native conformation challenging.

  • Solution: Use mild detergents (0.5-1% Triton X-100 or n-dodecyl-β-D-maltopyranoside) for solubilization, and include stabilizing agents (glycerol 5-50%) in buffers .

  • Alternatively, engineer truncated versions lacking membrane-interacting domains for solution studies.

• Maintaining Heme-Protein Covalent Bond:

  • The cysteine-heme covalent bond in C. necator CcmE (differs from histidine-heme in E. coli) is sensitive to reducing conditions.

  • Solution: Avoid strong reducing agents (DTT, β-mercaptoethanol) during purification; use argon-purged buffers to prevent oxidative damage.

  • For spectroscopic studies, use anaerobic cuvettes or rapid analysis after sample preparation.

• Spectroscopic Analysis Challenges:

  • The unique cysteine-heme bond creates distinct spectroscopic properties from the well-characterized histidine-heme bond.

  • Solution: Develop specialized reference spectra for C. necator CcmE; use multiple complementary techniques:

    • UV-visible spectroscopy for basic characterization

    • Resonance Raman spectroscopy to probe the heme-cysteine bond

    • NMR spectroscopy for protein-heme interaction sites

• Transient Interaction Analysis:

  • The interactions between CcmE and partner proteins (CcmC, CcmF) are often transient.

  • Alternatively, employ FRET-based assays with fluorescently labeled proteins for real-time interaction studies.

• Heme Transfer Kinetics:

  • Measuring the kinetics of heme transfer from CcmE to cytochrome c is technically challenging.

  • Solution: Develop a reconstituted system using:

    • Purified holo-CcmE

    • Apo-cytochrome c substrate

    • CcmF in nanodiscs or liposomes

    • Monitor transfer using stopped-flow spectroscopy with multiwavelength detection

• Structural Analysis:

  • The membrane association and dynamic nature of CcmE complexes makes structural determination difficult.

  • Solution: Use cryo-EM for the CcmABCE complex; for CcmE alone, use NMR with isotope labeling (15N, 13C) combined with selective heme labeling to map interaction surfaces .

These methodological approaches can overcome the technical challenges in studying the unique CcmE-heme interactions in C. necator, particularly the cysteine-based heme binding mechanism that differs from model organisms .

How can C. necator growth conditions be optimized for studying cytochrome c biogenesis?

Optimizing growth conditions for C. necator to study cytochrome c biogenesis requires careful consideration of media composition, culture parameters, and growth regimes:

• Media Composition Optimization:
  Based on statistical design of experiments (DOE), the following components significantly impact C. necator growth:

  Component	Optimal Concentration	Impact on Growth (t-value)	Notes for Cytochrome Studies
  Fructose	0.5-2.0 g/L	>1.65 (significant)	Primary carbon source for balanced growth
  Na2HPO4	4.18 g/L	>1.65 (significant)	Buffer maintenance
  Trace Elements	Standard mix	>1.65 (significant)	Essential for cytochrome formation
  CaCl2	0.4 mg/L	>1.65 (significant)	Cell membrane stability
  Copper	0.07 mg/L	Interacts with histidine	Critical for cytochrome oxidases
  Iron	1.6 mg/L	Not independently significant	Essential for heme synthesis - increase to 5-10 mg/L for cytochrome studies

  Note: For studying cytochrome c biogenesis specifically, increase iron concentration to 5-10 mg/L and ensure copper is properly balanced with histidine to avoid toxicity .

• Culture Conditions for Different Growth Modes:

  Growth Mode	Carbon Source	O2 Requirement	Temperature	Agitation	Notes
  Heterotrophic	Fructose (0.5 g/L)	21% (normal air)	30°C	180 RPM	Standard condition for lab studies
  Formatotrophic	Formic acid (1.5 g/L, pH-neutralized)	21% (normal air)	30°C	200 RPM	Higher agitation for better gas exchange
  Lithoautotrophic	H2/CO2/O2 (8:1:1)	10% O2 (safety!)	30°C	250 RPM	Special gas-tight vessels required
  Nitrate respiration	Fructose (0.5 g/L) + KNO3 (1 g/L)	Anaerobic	30°C	100 RPM	Sealed vessels with N2 headspace

• Bioreactor Setup for Controlled Studies:

  • Use 8-tube MC-1000-OD bioreactors (or similar) for parallel condition testing.

  • Implement chemostat cultivation at defined dilution rates (D = 0.1-0.3 h−1).

  • Bubble cultures with air at 12.5 mL/min for standard aerobic conditions.

  • Monitor OD720nm and OD680nm every 15 minutes for growth tracking.

  • For proteomics, sample after five retention times of continuous growth .

• Oxygen-Limited Growth for Enhanced Cytochrome Expression:

  • Microaerobic conditions often enhance cytochrome c expression.

  • Use deep-well plates or flasks filled to 60-70% volume with reduced agitation.

  • Monitor dissolved oxygen with optical sensors to maintain 2-5% saturation.

  • Add KNO3 (0.5-1 g/L) as alternative electron acceptor to further induce specific cytochromes .

These optimized conditions provide a methodological framework for studying cytochrome c biogenesis in C. necator across different growth regimes, enabling comprehensive analysis of CcmE function in various metabolic contexts.

How can recombinant CcmE be used to enhance electron transport efficiency in engineered C. necator strains?

Recombinant CcmE can be strategically employed to enhance electron transport efficiency in engineered C. necator strains through several approaches:

• Optimized CcmE Expression:

  • Controlled overexpression of CcmE using inducible promoters (such as rhamnose-inducible systems) can increase the rate of cytochrome c maturation.

  • This approach prevents bottlenecks in cytochrome c maturation during high metabolic demand .

• Engineered CcmE Variants:

  • Develop CcmE variants with enhanced heme binding and transfer capabilities through targeted mutations:

    • Optimize the cysteine-containing heme-binding motif (CXXXY)

    • Enhance interaction interfaces with CcmC and CcmF

  • Expression of these optimized variants can lead to more efficient electron transport chain assembly .

• Balanced Cytochrome c Biogenesis Pathway:

  • Co-express the complete CcmABCEF system in balanced ratios to prevent individual component limitations.

  • Experimental design:

    • Use polycistronic constructs with optimized spacing between genes

    • Employ multiple orthogonal inducible promoters for component-level control

    • Monitor respiratory capacity through oxygen consumption measurements

  • This approach ensures all components of the cytochrome c maturation pathway are available at optimal ratios .

• Substrate-Specific Optimization:

  • Tailor CcmE expression levels based on the substrate and electron transport complexes utilized:

  Growth Substrate	Key Respiratory Complexes	CcmE Optimization Strategy
  Formate	bc1 (QcrABC), cbb3 (CcoGNOP)	High CcmE expression coordinated with increased CcoGNOP
  Fructose	bc1, aa3 (CtaABCDEG)	Moderate CcmE expression with CtaABCDEG enhancement
  H2/CO2	bc1, mixed terminal oxidases	Balanced CcmE expression with hydrogenase coordination

  • This substrate-specific tuning matches cytochrome c production to actual metabolic needs .

• Synthetic Cytochrome c Variants:

  • Engineer specialized cytochrome c proteins with optimized redox properties for specific applications.

  • Use the enhanced CcmE system to efficiently mature these custom cytochromes.

  • Applications include improved electron transfer to terminal oxidases or artificial electron acceptors.

• Integration with Genome Streamlining:

  • Remove redundant respiratory complexes that are not utilized under specific conditions.

  • Redirect the saved protein resources toward optimized cytochrome c biogenesis.

  • This approach addresses the significant protein cost (up to 43% of proteome may be non-utilized) inherent in C. necator's metabolic versatility .

These strategies for CcmE optimization could significantly enhance electron transport efficiency in engineered C. necator strains, with applications in bioproduction, bioremediation, and bioelectrochemical systems .

What insights from C. necator CcmE studies can be applied to understanding cytochrome c biogenesis across bacterial species?

Studies of C. necator CcmE provide several valuable insights that can be applied to understanding cytochrome c biogenesis across bacterial species:

• Alternative Heme-Binding Mechanisms:

  • C. necator utilizes a cysteine residue (in CXXXY motif) for heme binding instead of the histidine (in HXXXY motif) found in E. coli.

  • This demonstrates functional convergence despite different biochemical mechanisms.

  • Implication: Researchers should examine non-canonical heme-binding residues when studying uncharacterized cytochrome c biogenesis systems .

• Streamlined System I Pathways:

  • C. necator lacks several components (CcmD, CcmH, CcmI) that are essential in model organisms like E. coli.

  • This reveals that the core process can function with fewer components than previously thought.

  • Provides a framework for identifying minimal requirements across diverse bacterial species .

• Evolutionary Patterns in Cytochrome c Biogenesis:

  • The distribution of different CcmE variants (histidine vs. cysteine-based) suggests multiple independent horizontal gene transfer events.

  • This pattern is observed across bacteria and archaea, indicating convergent evolution of similar functions.

  • This approach can reveal how cytochrome c biogenesis systems evolved across microbial lineages .

• Functional Adaptations to Energy Metabolism:

  • C. necator shows condition-specific utilization of different respiratory complexes.

  • Similar adaptations likely exist in other metabolically versatile bacteria.

  • This reveals how cytochrome c biogenesis is integrated with broader energy metabolism strategies .

• CcmE-CcmC Interaction Framework:

  • The identified interaction residues between CcmE and CcmC in C. necator (Arg73, Asp101, Glu105 on CcmE; Asp47, Gln50, Arg55 on CcmC) provide a template for understanding these interactions in other species.

  • This approach can reveal conserved mechanisms despite sequence divergence .

• Protein Resource Allocation Principles:

  • C. necator maintains significant expression of cytochrome c biogenesis genes even when not fully utilized.

  • This "readiness" strategy likely applies to other bacteria that encounter variable environments.

  • Proteomics and resource balance analysis approaches can be applied to other species to reveal similar patterns .

These insights from C. necator CcmE studies provide valuable frameworks for understanding cytochrome c biogenesis across bacterial species, particularly for organisms with metabolic versatility and those with non-canonical biogenesis systems .

What are the most promising future research directions for understanding CcmE function in C. necator?

Several promising future research directions can advance our understanding of CcmE function in C. necator:

• Structural Characterization of CcmE-Heme Complexes:

  • Determine high-resolution structures of C. necator CcmE with heme bound via the cysteine residue.

  • Compare with histidine-binding CcmE structures to understand mechanistic differences.

  • Apply techniques like cryo-EM for the membrane-associated CcmABCE complex.

  • This would provide unprecedented insights into the unique cysteine-based heme binding mechanism .

• Real-time Heme Transfer Kinetics:

  • Develop assays to monitor heme transfer from CcmE to cytochrome c in real-time.

  • Compare transfer rates between cysteine-based and histidine-based systems.

  • This would clarify functional consequences of the alternative heme binding mechanism .

• Synthetic Biology Applications:

  • Engineer minimal cytochrome c biogenesis systems based on C. necator components.

  • Create hybrid systems combining elements from different organisms.

  • Develop artificial electron transport chains with novel properties.

  • Research focus:

    • Optimize cytochrome c biogenesis for biotechnological applications

    • Engineer strains with enhanced electron transport efficiency

    • Create systems for bioelectrochemical applications .

• Systems Biology Integration:

  • Develop comprehensive models of how CcmE function integrates with:

    • Central carbon metabolism

    • Respiratory chain function

    • Redox homeostasis

    • Stress responses

  • This would reveal how CcmE contributes to C. necator's metabolic versatility .

• CcmE Function Under Stress Conditions:

  • Investigate how CcmE performance changes under:

    • Oxidative stress

    • Nutrient limitation

    • Oxygen fluctuations

    • Temperature stress

  • Research questions:

    • Does CcmE function limit stress adaptation?

    • How is cytochrome c biogenesis regulated under stress?

    • Does the cysteine-based mechanism provide advantages in specific conditions?

• Comparative Study Across Cupriavidus Species:

  • Examine CcmE variation across related species:

    • C. necator

    • C. taiwanensis

    • C. metallidurans

  • Focus on:

    • Sequence conservation

    • Heme-binding mechanism

    • Integration with metabolism

    • Functional adaptations to different ecological niches

  • This would provide evolutionary context for CcmE function in the genus .

• Heterologous Expression Systems Development:

  • Create optimized systems for expressing C. necator cytochromes in model organisms.

  • Utilize the knowledge of CcmE function to improve expression.

  • Applications:

    • Structural studies of difficult-to-produce cytochromes

    • Biochemical characterization of novel electron transport components

    • Production of specialized cytochromes for biotechnology .

These research directions would significantly advance our understanding of CcmE function in C. necator and provide valuable insights for both fundamental science and biotechnological applications .