Recombinant Pseudomonas putida Ferrochelatase (hemH)

What is Pseudomonas putida Ferrochelatase (hemH) and what is its biological function?

Pseudomonas putida Ferrochelatase (hemH) is an enzyme that catalyzes the terminal step in heme biosynthesis, inserting ferrous iron into protoporphyrin IX to form protoheme (heme B). It plays a critical role in iron metabolism and utilization in P. putida. The enzyme is also known as protoheme ferro-lyase (EC 4.99.1.1) and has a molecular weight of approximately 42.9 kDa .

In P. putida, hemH functions within a complex network of iron acquisition and porphyrin metabolism systems that are essential for various cellular processes, including respiration, oxidative stress response, and metal homeostasis . The enzyme's activity is particularly important given P. putida's environmental adaptability to diverse niches, including heavy metal-contaminated soils.

What expression systems are optimal for producing recombinant P. putida Ferrochelatase?

Multiple expression systems have been successfully employed for recombinant P. putida Ferrochelatase production:

E. coli expression systems are most commonly used due to their simplicity and high yields. For P. putida Ferrochelatase, expressing with an N-terminal His-tag in E. coli has been reported with purity levels of ≥74% and specific activity of ≥35 μmol/min/μg .

When expressing in E. coli, researchers should consider using BL21(DE3) or similar strains that lack certain proteases and provide tight control of expression, especially if using T7 promoter-based systems .

What are the optimized purification protocols for obtaining high-purity recombinant hemH?

A standard purification protocol for His-tagged P. putida Ferrochelatase typically involves:

• Cell lysis: Sonication or pressure-based disruption in buffer containing 40 mM Tris-HCl, pH 8.0, 110 mM NaCl, 2.2 mM KCl, with protease inhibitors.

• Initial clarification: Centrifugation at 10,000-15,000 × g for 30 minutes to remove cell debris.

• IMAC purification: Application of clarified lysate to Ni-NTA or similar affinity resin, washing with increasing imidazole concentrations (20-50 mM), and elution with high imidazole (200-300 mM).

• Further purification: Size exclusion chromatography using Superdex 75 or 200 columns for higher purity requirements.

• Storage: Final formulation in 40 mM Tris-HCl, pH 8.0, 110 mM NaCl, 2.2 mM KCl, 200 mM imidazole, and 20% glycerol for stability during freezing .

This protocol typically yields protein with ≥85% purity as assessed by SDS-PAGE . For applications requiring higher purity, additional ion exchange chromatography steps may be employed.

How is the enzymatic activity of P. putida Ferrochelatase measured in laboratory settings?

The enzymatic activity of P. putida Ferrochelatase can be measured through several established assays:

• Spectrophotometric assay: The most common method monitors the decrease in absorbance at 408 nm as protoporphyrin IX is converted to heme. The reaction contains:

  • 50-100 μM protoporphyrin IX

  • 50-100 μM ferrous ammonium sulfate (in 0.1% HCl to prevent oxidation)

  • 100 mM Tris-HCl buffer (pH 8.0)

  • 1-5 μg purified enzyme

  • Optional: 0.5% Tween 80 to solubilize the substrate

• Fluorometric assay: Measures the decrease in fluorescence of protoporphyrin IX (excitation 410 nm, emission 630 nm).

• HPLC analysis: Quantifies the formation of heme through reverse-phase HPLC.

For standardized activity reporting, one unit of enzyme activity is typically defined as the amount of enzyme that catalyzes the formation of 1 μmol of heme per minute under defined conditions. Commercial preparations typically have a specific activity of ≥35 μmol/min/μg .

What factors affect the stability and activity of recombinant P. putida Ferrochelatase?

Multiple factors influence enzyme stability and activity:

Enzyme stability is significantly enhanced by the addition of glycerol (20%) for long-term storage. Repeated freeze-thaw cycles should be avoided; working aliquots can be stored at 4°C for up to one week .

How does P. putida Ferrochelatase contribute to metal homeostasis in the bacterial cell?

P. putida Ferrochelatase plays a crucial role in iron utilization and homeostasis through several mechanisms:

• Iron incorporation: As the terminal enzyme in heme biosynthesis, it incorporates iron into protoporphyrin IX, making it a key consumer of the cellular iron pool.

• Integration with iron acquisition systems: The activity of hemH is coordinated with siderophore-based iron acquisition systems. P. putida contains multiple systems for iron uptake, including pyoverdine production and other siderophores .

• Metal sensing: Evidence suggests that hemH activity may be linked to metal sensing mechanisms, allowing cells to modulate iron utilization based on environmental availability.

The genome of P. putida encodes an unexpected capacity to tolerate heavy metals and metalloids, with 61 open reading frames likely involved in metal tolerance or homeostasis. These include systems for arsenic, chromate, divalent cations, monovalent cations, copper chelation, metallothionein for metal binding, and ABC transporters for essential metals . The hemH protein functions within this complex network of metal homeostasis systems.

What is the relationship between P. putida Ferrochelatase activity and oxidative stress response?

P. putida Ferrochelatase's role in oxidative stress response includes:

• Heme provision for protective enzymes: The enzyme supplies heme for incorporation into catalases and peroxidases that detoxify reactive oxygen species.

• Prevention of pro-oxidant accumulation: By efficiently converting protoporphyrin IX to heme, hemH prevents the accumulation of free porphyrins that can generate reactive oxygen species when exposed to light.

• Iron sequestration: Through iron incorporation into heme, hemH contributes to limiting free iron that could otherwise participate in Fenton reactions.

Research with related Pseudomonas species indicates that mutations affecting CcmC (involved in cytochrome c biogenesis) lead to reduced heme biosynthesis and increased susceptibility to oxidative stress . Similar connections likely exist for defects in hemH function, as both ultimately affect the cellular heme pool.

What genome editing approaches are effective for studying hemH function in P. putida?

Several genome editing approaches have been successfully applied to P. putida and could be used to study hemH function:

• CRISPR/Cas9-based systems: Modified CRISPR/Cas9n systems integrated with λ-Red recombination have been developed specifically for P. putida, allowing for precise genome editing with high efficiency . This approach maintains cell viability and genetic stability while increasing mutation efficiency.

• I-SceI-mediated recombination: A straightforward one-plasmid system for efficient genome editing in P. putida KT2440 has been demonstrated, which could be applied to hemH studies .

• Rec2 recombinase with mutLE36K: This system enables high-efficiency multi-site genomic editing of P. putida through ssDNA recombineering, achieving mutation frequencies of up to 10% for single-site mutations .

When studying hemH, researchers should consider using scarless deletion methods to avoid polar effects on neighboring genes, as hemH may be part of operons related to heme biosynthesis or iron metabolism.

How can recombinant hemH be integrated into P. putida for functional studies?

For functional studies requiring the integration of recombinant hemH variants into the P. putida genome:

• Chromosomal integration strategies:

  • Use of suicide vectors containing homology regions flanking the target integration site

  • CRISPR/Cas9-assisted homologous recombination for precise integration

  • Selection marker-based strategies using pyrF as both positive and negative selection

• Expression control options:

  • Native promoter replacement for physiological expression levels

  • Inducible promoters such as rhamnose-inducible (PrhaB) or arabinose-inducible (PBAD) systems

  • Constitutive promoters of varying strengths for stable expression

• Tagging considerations:

  • C-terminal tags generally have less impact on enzyme function than N-terminal tags

  • GFP fusions can be used to study localization

  • Epitope tags (FLAG, HA) for immunodetection with minimal functional interference

The choice between these approaches depends on the specific research questions. For complementation studies, expression from the native locus using the native promoter is preferable, while mechanistic studies might benefit from controlled expression using inducible systems .

How can P. putida Ferrochelatase be utilized in metabolic engineering applications?

P. putida Ferrochelatase represents a potential target for metabolic engineering in several contexts:

• Enhancement of heme-dependent pathways: Modulating hemH expression could increase the capacity of P. putida for processes requiring heme-containing enzymes, such as cytochrome P450-dependent reactions or peroxidase-based bioremediation.

• Integration with aromatic compound metabolism: P. putida is known for its ability to metabolize aromatic hydrocarbons . Engineering connections between heme biosynthesis and aromatic catabolism could create more efficient bioremediation strains.

• Polyhydroxyalkanoate (PHA) production: P. putida has been engineered for PHA production from various substrates, including aromatic compounds . Optimizing iron and heme metabolism through hemH engineering could potentially enhance energy metabolism and carbon flux toward PHA biosynthesis.

Research has demonstrated that stepwise genetic engineering of P. putida enables high-titer production of various compounds, reaching levels of 100 mg - 1 g/L for certain products . Similar engineering approaches targeting hemH and related pathways could yield strains with enhanced capabilities for specific biotechnological applications.

What is known about the interplay between hemH and rare earth element utilization in P. putida?

Recent research has uncovered fascinating connections between rare earth elements (REEs) and P. putida metabolism:

• REE-dependent growth: P. putida KT2440 expresses a REE-dependent and pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase called PedH, which is vital for growth on alcoholic volatiles .

• REE-switch mechanism: The production of PedH and its Ca²⁺-dependent counterpart PedE is inversely regulated in response to lanthanide bioavailability through a mechanism termed the REE-switch .

• Influence of metal availability: Copper, zinc, and particularly iron availability influence this regulation in a pyoverdine-independent manner by increasing the minimal lanthanide concentration required for the REE-switch by several orders of magnitude .

The connection between hemH and REE metabolism likely involves complex interactions within the metal homeostasis network. As hemH requires ferrous iron for its catalytic activity, changes in iron availability due to REE presence could affect hemH function and consequently heme biosynthesis. This represents an emerging area of research with potential implications for understanding bacterial adaptation to metal-rich environments .

What strategies can address low activity of recombinant P. putida Ferrochelatase?

When encountering low activity of recombinant hemH, researchers can implement several troubleshooting strategies:

For activity measurements, ensure that protoporphyrin IX is properly solubilized and that ferrous iron remains reduced throughout the assay. Consider adding reducing agents like 0.5-1 mM DTT, but be aware that these can also affect iron availability for the reaction.

How can researchers effectively study hemH in the context of P. putida's complex metal homeostasis network?

Studying hemH within P. putida's complex metal homeostasis network requires multifaceted approaches:

• Combinatorial gene deletions: Create strains with deletions in multiple metal homeostasis genes to identify functional interactions with hemH. The genome of P. putida encodes 61 open reading frames likely involved in metal tolerance or homeostasis .

• Metal-defined growth conditions: Use chemically defined media with precise control of metal availability. Studying growth under varying concentrations of iron, copper, zinc, and rare earth elements can reveal the interconnections between different metal utilization pathways .

• Transcriptomic and proteomic profiling: Apply RNA-Seq and proteomics to identify co-regulated genes under different metal availability conditions.

• In vivo activity probes: Develop fluorescent or colorimetric reporters that respond to hemH activity or heme levels to monitor the system dynamically in living cells.

• Integration with metabolic models: Incorporate hemH and related pathways into genome-scale metabolic models of P. putida to predict systemic effects of alterations. Advanced models with proteomic and kinetic constraints have been developed for P. putida and can be extended to include detailed metal homeostasis pathways .

When designing such studies, researchers should be aware that the REE-switch in P. putida is influenced by environmental conditions, with critical lanthanide concentrations required to support growth varying dramatically depending on the medium used, ranging from 5 nM up to 10 μM .