Recombinant Photobacterium profundum Cytochrome c-type biogenesis protein CcmE (ccmE), partial

What is the function of CcmE in Photobacterium profundum?

CcmE in P. profundum serves as a heme chaperone protein essential for cytochrome c biogenesis. It functions by:

• Taking heme from CcmC in the membrane-associated CcmABCD complex

• Forming a covalent bond with heme to create holo-CcmE

• Transporting heme across the cytoplasmic membrane

• Serving as a heme reservoir when bound to CcmC

• Ultimately transferring heme to apocytochrome c via the CcmF/H complex (cytochrome c synthetase)

In P. profundum, CcmE is particularly significant because cytochrome c proteins are crucial for electron transport in respiration systems that function under high hydrostatic pressure conditions .

How does the cytochrome c biogenesis pathway in P. profundum compare to other bacterial species?

The cytochrome c biogenesis in P. profundum follows the System I pathway (also found in other proteobacteria) with several key features:

P. profundum's CcmE likely has adaptations that allow it to function efficiently under high pressure, whereas E. coli's system is optimized for atmospheric pressure .

What is the relationship between pressure adaptation and cytochrome c biogenesis in P. profundum?

P. profundum strain SS9's pressure adaptation involves several mechanisms that potentially affect CcmE function and cytochrome c biogenesis:

• Differential protein expression: Proteomic analysis shows that P. profundum differentially expresses proteins involved in key metabolic pathways at high versus atmospheric pressure

• Respiratory chain modulation: Proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure, whereas glycolysis/gluconeogenesis pathway proteins are up-regulated at high pressure

• Membrane composition changes: At low temperature and high pressure, P. profundum alters its fatty acid composition in cell membranes, which could affect membrane protein function including the Ccm system

• Stress response regulation: Several stress response genes (htpG, dnaK, dnaJ, groEL) are up-regulated at atmospheric pressure, which may indirectly affect protein folding and stability of components in the cytochrome c biogenesis pathway

These adaptations suggest that the cytochrome c biogenesis pathway, including CcmE function, may be optimized to maintain efficient electron transport under high-pressure conditions .

How does the CcmE-mediated heme transport mechanism function under different pressure conditions in P. profundum?

The heme transport mechanism mediated by CcmE in P. profundum likely exhibits pressure-dependent modifications:

At High Pressure (28 MPa):

• Enhanced stability of CcmE-heme interactions possibly due to volume-reducing conformational changes

• Potential alteration in ATP-dependent release kinetics from the CcmABCD complex

• Modified protein-protein interactions that may optimize heme transfer efficiency

• Possible altered water accessibility to the heme binding pocket

At Atmospheric Pressure (0.1 MPa):

• Different conformational equilibrium that may affect heme binding capacity

• Altered interaction dynamics with CcmC and other components of the complex

• Potentially less efficient heme transfer to apocytochrome c

Research using pressure-perturbation studies with cytochrome P450 from P. profundum has shown that high pressure can induce conformational changes that affect water accessibility to the heme pocket, which could be a common feature in heme-binding proteins from this organism. The transition to states with confined water accessibility may represent an evolutionary adaptation to function under high hydrostatic pressure .

Current data suggests that pressure-adapted proteins in P. profundum may have evolved unique structural features that allow them to maintain functionality at deep-sea pressures, and CcmE likely possesses similar adaptations to ensure efficient cytochrome c biogenesis under these conditions .

What are the structural and functional implications of the absence of CcmD homologs in some organisms compared to P. profundum?

The absence of CcmD in some organisms (particularly archaea) compared to its presence in P. profundum has significant implications:

Functional Implications:

• In P. profundum and E. coli, CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex

• Studies show that CcmD is a component of the CcmABC ATP-binding cassette transporter complex in E. coli

• CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE or for interaction of CcmE with CcmABC

Alternative Mechanisms in CcmD-lacking Organisms:

• Archaeal genomes that lack CcmD, CcmH, and CcmI still produce functional cytochrome c, suggesting alternative release mechanisms

• Research with Methanosarcina acetivorans demonstrated that a streamlined version of Ccm machinery (only CcmABCEF) is sufficient for cytochrome c biogenesis in archaea

• This suggests that CcmD's function in facilitating holo-CcmE release may be integrated into other components in some organisms

Research Implications:

• Comparative studies between P. profundum CcmE and archaeal CcmE could reveal alternative mechanisms for holo-CcmE release

• The topology of CcmD (N-terminus outside, C-terminus inside, with one transmembrane domain) in P. profundum may differ from functional equivalents in other organisms

• Understanding these differences could provide insights into the evolution of cytochrome c biogenesis pathways and their adaptation to different environmental conditions

These findings highlight how diverse organisms have evolved different solutions to the same biochemical challenge, with P. profundum utilizing the complete CcmABCDEFGH system while some archaea function with a reduced set of components .

How can mutations in P. profundum CcmE be used to investigate the effects of pressure on heme binding and release?

Strategic mutations in P. profundum CcmE can provide valuable insights into pressure effects on heme binding and release through the following approaches:

Key Mutation Strategies:

• Mutations at the heme-binding site (histidine residue) to study pressure effects on covalent attachment

• Alterations in residues interacting with CcmC to investigate pressure effects on complex formation

• Modifications of residues involved in CcmF/H interaction to examine heme transfer under pressure

• Introduction of pressure-sensing reporter groups at strategic positions

Methodological Approaches:

• Site-directed mutagenesis to create specific CcmE variants

• High-pressure spectroscopy to monitor heme binding under variable pressure conditions

• Pressure-perturbation studies to analyze conformational changes

• In vitro reconstitution assays to measure heme transfer efficiency at different pressures

Expected Insights:

• Identification of pressure-sensitive regions within the CcmE structure

• Understanding of how hydrostatic pressure affects the thermodynamics and kinetics of heme binding

• Elucidation of pressure-adaptive features in P. profundum CcmE compared to homologs from non-piezophilic bacteria

• Determination of how pressure affects the interaction network between CcmE and other Ccm proteins

Research with P. profundum cytochrome P450 has shown that pressure can affect conformational equilibria and water accessibility to the heme pocket, suggesting similar mechanisms may apply to CcmE . By comparing wild-type and mutant CcmE behavior under varying pressure conditions, researchers can map the molecular basis of pressure adaptation in this critical heme chaperone protein.

What are the optimal protocols for expressing and purifying recombinant P. profundum CcmE?

Expression System Optimization:

Purification Protocol:

• Harvest cells by centrifugation at 800×g for a moderate duration (10 min) to preserve protein integrity

• Resuspend in lysis buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors

• Lyse cells using sonication or French press

• Separate membrane and soluble fractions by ultracentrifugation (100,000×g, 1 hour)

• Solubilize membrane fraction with 1% n-dodecyl-β-D-maltoside if CcmE is membrane-associated

• Purify using Ni-NTA affinity chromatography with imidazole gradient elution

• Further purify by size exclusion chromatography

Special Considerations:

• Addition of 5-aminolevulinic acid (heme precursor) to growth medium may enhance holo-CcmE formation

• Consider pressure treatment of cell lysate if investigating pressure-dependent conformations

• Verify heme incorporation using absorption spectroscopy (Soret band monitoring)

• For membrane-bound forms, anti-Strep affinity columns have been successfully used

This protocol is based on methods that have successfully been used to express and purify other P. profundum proteins, with modifications specific to CcmE based on its biochemical properties .

How can the functionality of recombinant P. profundum CcmE be assessed in vitro and in vivo?

In Vitro Functionality Assays:

• Heme Binding Assessment:

  • UV-visible spectroscopy to measure the characteristic Soret band (~400 nm) and α/β bands (500-600 nm)

  • Pyridine hemochrome assay to quantify covalently bound heme

  • Resonance Raman spectroscopy to analyze heme-protein interactions

• Thermal and Pressure Stability:

  • Differential scanning calorimetry to determine thermal stability

  • High-pressure spectroscopy to monitor conformational changes under pressure

  • Circular dichroism spectroscopy to assess secondary structure integrity

• Heme Transfer Activity:

  • In vitro reconstitution assay with purified CcmF/H to measure heme transfer rate

  • Stopped-flow kinetics to determine the rate constants for heme binding and release

  • Fluorescence resonance energy transfer (FRET) to monitor protein-protein interactions

In Vivo Functionality Assays:

• Complementation Studies:

  • Transformation of recombinant CcmE into ΔccmE knockout strains

  • Assessment of cytochrome c levels using heme staining of SDS-PAGE gels

  • Measurement of cellular respiration rates using oxygen consumption assays

• Pressure-Dependent Functionality:

  • Growth assays under varying pressure conditions (0.1-70 MPa)

  • Analysis of cytochrome c content in cells grown under different pressures

  • Immunoblotting with anti-FLAG antibodies for tagged-CcmE detection

• Protein-Protein Interaction Assessment:

  • Co-immunoprecipitation with other Ccm components

  • Bacterial two-hybrid assays to identify interaction partners

  • In vivo crosslinking followed by mass spectrometry

Representative Data Example:
A successful complementation assay would show restoration of cytochrome c production in a ΔccmE strain transformed with functional recombinant CcmE, as evidenced by:

• Detection of heme-bound CcmE by heme staining the enriched membrane fraction

• Restoration of respiratory capacity measured by oxygen consumption

• Growth recovery under conditions requiring cytochrome c function

Research with M. acetivorans has demonstrated successful detection of tagged-CcmE in membrane fractions using anti-Strep affinity columns and immunoblotting with anti-FLAG antibodies, which could be adapted for P. profundum CcmE .

What high-pressure experimental systems are recommended for studying P. profundum CcmE function?

Several specialized high-pressure experimental systems can be used to study P. profundum CcmE function:

High-Pressure Growth Systems:

• Pressure Vessels for Batch Culture:

  • Stainless-steel pressure vessels capable of maintaining 28 MPa (optimal for P. profundum SS9)

  • Temperature control at 15-17°C

  • Anaerobic culturing capability with sealed polyethylene transfer pipettes

  • Application: Growing cells for CcmE expression under native pressure conditions

• Continuous High-Pressure Bioreactors:

  • Systems that allow sampling during growth without depressurization

  • Pressure range of 0.1-70 MPa to cover the entire growth range of P. profundum

  • Application: Studying pressure-dependent regulation of CcmE expression

High-Pressure Biochemical Analysis Systems:

• High-Pressure Spectroscopy:

  • Pressure cells with optical windows for UV-visible, fluorescence, and CD spectroscopy

  • Pressure range: 0.1-100 MPa

  • Application: Monitoring CcmE-heme interactions under pressure

• High-Pressure Direct Visualization System (HPDS):

  • Used successfully to examine P. profundum motility under pressure

  • Allows microscopic observation of cells under pressure

  • Application: Studying localization of fluorescently tagged CcmE in living cells

• High-Pressure Stopped-Flow Apparatus:

  • For kinetic measurements of heme binding/release under pressure

  • Pressure range: 0.1-200 MPa

  • Application: Determining how pressure affects CcmE reaction rates

Experimental Design Considerations:

For high-pressure growth experiments with P. profundum, researchers have successfully used sealed polyethylene transfer pipettes placed in water-cooled pressure vessels at 28 MPa and 17°C . This approach has been validated in studies of P. profundum motility under pressure and could be adapted for investigating CcmE function.

How can interactions between CcmE and other components of the cytochrome c biogenesis system be studied in P. profundum?

Investigating the interactions between CcmE and other components of the cytochrome c biogenesis system in P. profundum requires specialized methods that account for membrane protein complexes and high-pressure adaptation:

Protein-Protein Interaction Methods:

• Co-Immunoprecipitation under Native Conditions:

  • Tag CcmE with epitope tags (His, FLAG, or Strep) as successfully done in M. acetivorans

  • Use cross-linking agents to capture transient interactions

  • Perform IP under varying pressure conditions after pressure treatment

  • Identify interaction partners by mass spectrometry

  • Example protocol: Anti-FLAG immunoblotting has been used successfully to detect tagged-CcmE in protein enriched from membrane fractions

• Bacterial Two-Hybrid System:

  • Adapt for psychrophilic growth at 15-17°C

  • Screen for interactions between CcmE and other Ccm proteins

  • Validate interactions in different pressure-growth backgrounds

• In Vitro Reconstitution:

  • Purify individual components (CcmA, CcmB, CcmC, CcmD, CcmE)

  • Reconstitute in liposomes with appropriate lipid composition

  • Measure complex formation and function under pressure

• Surface Plasmon Resonance:

  • Immobilize one component (e.g., CcmE)

  • Measure binding kinetics with other components under varying pressure conditions

  • Analyze pressure effects on association and dissociation rates

Localization and Dynamics Studies:

• Fluorescence Microscopy with Pressure Chambers:

  • Create fluorescent protein fusions (GFP-CcmE) in P. profundum

  • Visualize localization under pressure using specialized chambers

  • P. profundum has been successfully visualized expressing GFP

• FRET Analysis:

  • Create donor-acceptor pairs of Ccm components

  • Measure interaction dynamics in live cells under pressure

  • Quantify distance changes and conformational dynamics

Structural Analysis:

• Cryo-Electron Microscopy:

  • Isolate native CcmABCDE complexes from P. profundum

  • Determine structure under different pressure conditions

  • Compare with structures from non-piezophilic bacteria

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Map interaction surfaces between CcmE and other components

  • Identify pressure-sensitive regions in the protein complex

  • Determine conformational changes upon complex formation

Genetic Manipulation Approaches:

• Marker Exchange-Eviction Mutagenesis:

  • Create targeted mutations in interaction domains

  • Use the sacB-containing suicide vector method established for P. profundum

  • Assess effects on complex formation and function

  • This approach has been successfully used to create flagellin and motor protein mutants in P. profundum