Recombinant Herpetosiphon aurantiacus Ferrochelatase (hemH)

What is Herpetosiphon aurantiacus Ferrochelatase (hemH) and what is its primary function?

Herpetosiphon aurantiacus Ferrochelatase (hemH) is an enzyme that catalyzes the terminal step in heme biosynthesis, specifically the insertion of ferrous iron into protoporphyrin to produce heme. The recombinant form available for research has a Uniprot accession number of A9B546 and consists of 305 amino acids. This enzyme belongs to the larger family of ferrochelatases (EC 4.99.1.1), also known as heme synthase or protoheme ferro-lyase, which are present across various organisms .

Methodologically, the enzyme can be studied through activity assays monitoring either substrate consumption (protoporphyrin IX decrease) or product formation (heme increase) spectrophotometrically, as the conversion results in distinct spectral changes.

What are the optimal storage and handling conditions for recombinant Herpetosiphon aurantiacus Ferrochelatase?

For optimal preservation of enzyme activity, recombinant Herpetosiphon aurantiacus Ferrochelatase should be stored at -20°C for short-term use and at -20°C or -80°C for extended storage. Repeated freezing and thawing cycles should be avoided to prevent denaturation and activity loss. Working aliquots can be maintained at 4°C for up to one week .

For reconstitution, the manufacturer recommends briefly centrifuging the vial before opening and then reconstituting the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (typically 50%) is recommended for long-term storage stability .

What expression systems are used for producing recombinant Herpetosiphon aurantiacus Ferrochelatase?

Recombinant Herpetosiphon aurantiacus Ferrochelatase is typically expressed in E. coli expression systems. The commercially available protein described in the product datasheet is produced in E. coli with purity >85% as determined by SDS-PAGE .

When designing expression systems for research purposes, considerations should include:

• Codon optimization for E. coli

• Selection of appropriate fusion tags to enhance solubility and facilitate purification

• Growth conditions that maximize soluble protein yield

• Purification strategies that maintain enzyme activity

What are the recommended protocols for measuring Herpetosiphon aurantiacus Ferrochelatase activity?

Ferrochelatase activity can be measured through multiple complementary approaches:

For optimal results, researchers should consider:

• Using anaerobic conditions to prevent iron oxidation

• Including appropriate metal chelators to control free metal ion concentrations

• Using physiologically relevant pH (typically 7.5-8.0)

• Controlling temperature (typically 30-37°C)

• Including detergents or phospholipids to maintain enzyme stability

How can site-directed mutagenesis be used to investigate the catalytic mechanism of Herpetosiphon aurantiacus Ferrochelatase?

Site-directed mutagenesis provides a powerful approach to dissect the structure-function relationships in ferrochelatase. Based on studies of homologous enzymes, researchers can target key residues potentially involved in:

• Metal binding (e.g., M76): Mutations like M76A, M76L, or M76F can help determine the role of this residue in metal coordination and substrate specificity .

• Proton abstraction (e.g., E343): Mutations such as E343Q, E343D, or E343A can test the importance of this residue in acid-base catalysis .

• Metal sensing (peptide loop Q302-K304): Alanine scanning of these residues can reveal their roles in metal detection and enzyme conformational changes .

Methodologically, researchers should:

• Create single and potentially double/triple mutants

• Express and purify mutant proteins under identical conditions

• Perform comprehensive kinetic analyses (Km, kcat, pH profiles)

• Conduct metal binding studies to assess changes in affinity

• When possible, determine crystal structures of key mutants

What approaches can be used to determine metal binding properties of Herpetosiphon aurantiacus Ferrochelatase?

Understanding metal binding properties requires multiple complementary techniques:

These approaches can reveal critical information about how Herpetosiphon aurantiacus Ferrochelatase selects and positions metal ions for catalysis, informing both fundamental understanding and potential enzyme engineering efforts.

How does the active site architecture of Herpetosiphon aurantiacus Ferrochelatase compare to other bacterial ferrochelatases?

While the search results don't provide direct structural comparison, studies on ferrochelatase mechanisms indicate conserved elements across species. Comparing H. aurantiacus Ferrochelatase to other bacterial ferrochelatases reveals important insights:

• Active site residues: Key residues like M76 (involved in metal binding) and E343 (involved in proton abstraction) appear to be functionally conserved across bacterial ferrochelatases, suggesting a common catalytic mechanism .

• Metal sensing: The peptide loop composed of Q302, S303, and K304 that acts as a metal sensor in ferrochelatases appears to be a conserved feature, though the specific residues may vary between species .

Methodologically, researchers can perform:

• Sequence alignments using tools like CLUSTAL Omega

• Homology modeling if crystal structures are unavailable

• Superposition of active sites across different species

• Phylogenetic analysis to trace the evolution of key catalytic features

What is known about the substrate specificity of Herpetosiphon aurantiacus Ferrochelatase compared to ferrochelatases from other organisms?

The substrate specificity of ferrochelatases varies across organisms, though comprehensive comparative data for H. aurantiacus Ferrochelatase specifically is not available in the search results. Generally, ferrochelatases show specificity for:

• Porphyrin substrates: Most ferrochelatases preferentially use protoporphyrin IX but may accept modified porphyrins to varying degrees.

• Metal ions: Ferrochelatases typically show highest activity with Fe²⁺, though many can insert Zn²⁺, Co²⁺, and other divalent metals at lower rates.

To systematically investigate substrate specificity, researchers should:

• Test activity with various porphyrin substrates (protoporphyrin IX, mesoporphyrin, deuteroporphyrin)

• Evaluate insertion of different metal ions (Fe²⁺, Zn²⁺, Co²⁺, Ni²⁺, Cu²⁺)

• Determine kinetic parameters for each substrate-metal combination

• Compare results with literature data for ferrochelatases from other species

How does Herpetosiphon aurantiacus Ferrochelatase fit into the organism's metabolic network?

Herpetosiphon aurantiacus is a nonphototrophic, strictly aerobic, gliding bacterium belonging to the green nonsulfur bacteria phylum . Within this organism's metabolic network, ferrochelatase likely plays crucial roles:

• Terminal enzyme in heme biosynthesis: As in other organisms, H. aurantiacus Ferrochelatase catalyzes the final step in heme production, providing essential cofactors for cytochromes and other hemoproteins.

• Relation to the organism's unique features: H. aurantiacus has a deep orange pigmentation in its cytoplasmic membrane , suggesting active production of colored compounds potentially related to tetrapyrrole metabolism.

• Potential connections to other pathways: The search results indicate that H. aurantiacus produces various specialized metabolites, including a diterpene called herpetopanone , suggesting complex secondary metabolic networks that may interact with heme biosynthesis.

To fully map the metabolic context of ferrochelatase in H. aurantiacus, researchers should employ:

• Comparative genomics to identify co-localized genes

• Transcriptomics to identify co-regulated genes

• Metabolomics to detect related metabolites

• Gene disruption studies to assess physiological impact

What are the potential applications of recombinant Herpetosiphon aurantiacus Ferrochelatase in synthetic biology?

Recombinant Herpetosiphon aurantiacus Ferrochelatase has several potential applications in synthetic biology:

• Engineered heme biosynthesis: The enzyme could be used in heterologous hosts to produce heme or modified tetrapyrroles for various applications.

• Biocatalysis: Ferrochelatase can catalyze the insertion of various metals into porphyrins, potentially enabling the synthesis of novel metalloporphyrins for catalytic or sensing applications.

• Biosensors: The metal-insertion activity could be harnessed for the development of biosensors for metal ions or porphyrins.

• Synthetic pathway engineering: In combination with other tetrapyrrole biosynthesis enzymes, ferrochelatase could enable the construction of artificial pathways for specialized metalloporphyrin production.

• Photosynthesis research: Given that H. aurantiacus belongs to the green nonsulfur bacteria phylum , its ferrochelatase may have properties optimized for integration with photosynthetic systems, potentially offering advantages for synthetic photosystems.

How can the crystal structure of Herpetosiphon aurantiacus Ferrochelatase be determined, and what insights would it provide?

While the crystal structure of H. aurantiacus Ferrochelatase is not reported in the search results, determining this structure would provide valuable insights. Methodological approaches include:

• X-ray crystallography workflow:

  • High-yield expression and purification of recombinant protein

  • Crystallization screening with commercial kits

  • Optimization of crystallization conditions

  • Data collection at synchrotron radiation sources

  • Structure determination using molecular replacement with homologous structures

• Cryo-electron microscopy:

  • Sample preparation on grids

  • Data collection using high-end microscopes

  • Image processing and 3D reconstruction

  • Model building and refinement

• Target states for structural characterization:

  • Apo-enzyme

  • Enzyme-substrate complex

  • Enzyme-product complex

  • Enzyme with various metals bound

The structural data would provide insights into:

• Active site architecture and catalytic mechanism

• Metal binding sites and specificity determinants

• Substrate recognition features

• Conformational changes during catalysis

• Potential protein-protein interaction surfaces

What are the challenges in expressing and purifying active Herpetosiphon aurantiacus Ferrochelatase at high yields?

Producing high yields of active ferrochelatase presents several challenges that must be addressed through systematic optimization:

The commercial preparation achieves >85% purity by SDS-PAGE , suggesting effective purification strategies exist, though yields and specific activity are not provided.

How can isotope labeling approaches be applied to study the catalytic mechanism of Herpetosiphon aurantiacus Ferrochelatase?

Isotope labeling provides powerful tools for investigating enzyme mechanisms. For H. aurantiacus Ferrochelatase, several approaches are relevant:

• Deuterium labeling: Using deuterated substrates (e.g., protoporphyrin IX with deuterated pyrrole nitrogens) to investigate kinetic isotope effects can reveal if proton abstraction is rate-limiting.

• ¹⁸O labeling: Incorporating ¹⁸O into the reaction can help track oxygen atoms and determine if water molecules participate in the reaction.

• ¹⁵N labeling: Labeling the pyrrole nitrogens can help track protonation/deprotonation events during catalysis.

• ¹³C labeling: Site-specific ¹³C labeling of the enzyme through metabolic incorporation during expression can enable NMR studies of enzyme dynamics and substrate interactions.

• Metal isotope labeling: Using isotopes of iron (⁵⁴Fe, ⁵⁷Fe) can facilitate tracking of metal incorporation and potentially enable Mössbauer spectroscopy studies.

These approaches can be particularly powerful when combined with spectroscopic techniques like NMR, mass spectrometry, and vibrational spectroscopy to track isotope movement during catalysis.

What role might ferrochelatase play in the ecological interactions of Herpetosiphon aurantiacus in its natural environment?

Herpetosiphon aurantiacus is a predatory gliding bacterium , and ferrochelatase may contribute to its ecological interactions in several ways:

• Support for predatory behavior: As a predatory bacterium, H. aurantiacus likely requires cytochromes and other hemoproteins for energy metabolism to support its active hunting behavior. Ferrochelatase supplies the necessary heme cofactors for these proteins.

• Potential role in antibiotic production: Predatory bacteria often produce antibiotics to kill prey bacteria. If any of these involve heme or modified tetrapyrroles, ferrochelatase would be essential for their biosynthesis.

• Environmental adaptation: H. aurantiacus has a permeability barrier on its surface with a channel-forming protein , and hemoproteins may play roles in sensing or responding to environmental conditions through this barrier.

• Relation to pigmentation: The deep orange color of H. aurantiacus may involve tetrapyrrole derivatives, potentially linking ferrochelatase activity to pigment production.

• Interspecies competition: The ability to efficiently acquire and utilize iron through ferrochelatase activity may provide competitive advantages in iron-limited environments.

Research approaches to investigate these ecological roles could include comparative genomics across Herpetosiphon species, transcriptomics under different predatory conditions, and metabolomic analysis of tetrapyrrole derivatives in different ecological contexts.

What are the key unresolved questions about Herpetosiphon aurantiacus Ferrochelatase that warrant further investigation?

Despite the information available about Herpetosiphon aurantiacus Ferrochelatase, several critical questions remain unanswered:

• Structural features: The high-resolution crystal structure of H. aurantiacus Ferrochelatase has not been determined, leaving questions about its precise active site architecture and substrate binding mode.

• Catalytic mechanism: The specific roles of conserved residues like M76 and E343 in the H. aurantiacus enzyme need experimental validation.

• Substrate specificity: Comprehensive characterization of substrate preferences, including various porphyrins and metal ions, would provide insights into the enzyme's biological roles.

• Metabolic integration: The position of ferrochelatase in the organism's broader metabolic network, including potential connections to specialized metabolite production, remains to be fully mapped.

• Ecological significance: The role of ferrochelatase in supporting H. aurantiacus's predatory lifestyle and environmental adaptations represents an intriguing area for future investigation.

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and ecology, potentially yielding insights relevant to both fundamental understanding and biotechnological applications.

How can integrating computational and experimental approaches advance our understanding of Herpetosiphon aurantiacus Ferrochelatase?

Integrating computational and experimental methods offers a powerful strategy for investigating H. aurantiacus Ferrochelatase:

• Structure prediction and validation:

  • Use AlphaFold2 or RoseTTAFold to predict the enzyme's structure

  • Validate predictions with experimental techniques like HDX-MS or crosslinking

  • Design experiments to test structural hypotheses

• Mechanism modeling and testing:

  • Employ QM/MM simulations to model the catalytic mechanism

  • Use site-directed mutagenesis to test computational predictions

  • Refine models based on experimental results

• Substrate specificity engineering:

  • Use molecular docking to predict binding of alternative substrates

  • Design mutations to alter specificity based on computational insights

  • Test engineered variants experimentally

• Systems biology integration:

  • Model ferrochelatase in the context of whole-cell metabolism

  • Predict metabolic impacts of altered ferrochelatase activity

  • Validate predictions with metabolomics and transcriptomics