Recombinant Photobacterium profundum Hydroxylamine reductase (hcp), partial

What is the hybrid cluster protein (Hcp) in Photobacterium profundum?

Hybrid cluster protein (Hcp) in P. profundum is a redox enzyme that plays a crucial role in nitrogen metabolism. Originally termed the "prismane protein" due to its unique spectroscopic properties, Hcp contains distinctive iron-sulfur clusters, including a hybrid [4Fe-2S-2O] cluster . In P. profundum, as in other bacteria, the hcp gene typically forms an operon with NADH oxidoreductase (hcr), whose product catalyzes the reduction of Hcp in the presence of NADH .

Hcp is particularly induced during conditions of nitrite or nitrate stress, highlighting its importance in nitrogen metabolism pathways . The primary function identified for bacterial Hcps is hydroxylamine reductase activity, which converts hydroxylamine (NH₂OH) to ammonia (NH₃) and water, serving as a detoxification mechanism for reactive nitrogen intermediates .

What is hydroxylamine reductase activity and how is it measured experimentally?

Hydroxylamine reductase activity refers to the enzymatic conversion of hydroxylamine (NH₂OH) to ammonia (NH₃) and water. The reaction can be represented as:

NH₂OH + 3e⁻ + 3H⁺ → NH₃ + H₂O

This activity can be measured through several methodological approaches:

• Spectrophotometric assay: Monitoring the oxidation of the electron donor (typically reduced methyl viologen) as hydroxylamine is reduced .

• Ammonia quantification: Following a hydroxylamine reduction reaction (approximately 10 minutes), add saturated sodium carbonate (Na₂CO₃) and use a microdiffusion stopper with sulfuric acid (H₂SO₄) to capture released ammonia. After a 3-hour diffusion period, the stopper is removed and the captured ammonia is quantified using phenol reagent and alkaline hypochlorite, measuring absorbance spectrophotometrically .

• pH-dependent activity measurements: Using a mixed buffer system (containing MES, MOPS, HEPES, Tris-HCl, and CHES) to maintain desired pH levels while determining hydroxylamine reduction rates .

For P. profundum Hcp specifically, these methods might need modification to account for its psychrophilic and piezophilic adaptations.

How does pH affect hydroxylamine reductase activity in bacterial Hcps?

The pH significantly influences hydroxylamine reductase activity in bacterial Hcps. While specific data for P. profundum Hcp is not directly available in the search results, insights can be drawn from related studies.

For other bacterial Hcps, researchers have conducted pH-dependent rate studies across pH ranges from 6 to 10 using mixed buffer systems . The pH dependency pattern is critical for understanding the catalytic mechanism and physiological role of the enzyme.

Interestingly, P. profundum α-carbonic anhydrase (PprCA) exhibits unusual bimodal pH activity with peak activity at both acidic (pH 5) and alkaline (pH 11) conditions. At pH 5, it retains 88% of its maximum activity observed at pH 11 . This unusual pH profile may be an adaptation to varying environmental conditions in the deep sea.

If P. profundum Hcp exhibits similar adaptations, it could maintain significant activity across a broader pH range compared to mesophilic counterparts, providing metabolic versatility in changing deep-sea environments.

What is the relationship between Hcp and nitrogen metabolism?

Hcp plays a significant role in bacterial nitrogen metabolism, particularly in the detoxification of reactive nitrogen species. The enzyme is typically induced during conditions of nitrite or nitrate stress, indicating its importance in nitrogen homeostasis .

The primary function of Hcp is hydroxylamine reductase activity, converting the toxic intermediate hydroxylamine (NH₂OH) to non-toxic ammonia (NH₃) and water . This activity prevents accumulation of hydroxylamine, which can damage cellular components. The requirement of the hcp gene for in vivo hydroxylamine reduction has been demonstrated in organisms like Rhodobacter capsulatus E1F1 .

In the regulatory network controlling nitrogen oxide metabolism, Hcp expression is regulated by transcription factors responding to nitrogen availability and redox conditions. In many bacteria, the hcp gene is part of the NsrR regulon, which responds to nitrosative stress . Additionally, in some organisms like Desulfovibrio species, Hcp regulation is linked to the sulfate reduction pathway, suggesting crosstalk between nitrogen and sulfur metabolism .

How does oxygen exposure affect hydroxylamine reductase activity?

Oxygen exposure significantly impacts hydroxylamine reductase activity of Hcp. Experimental data reveals a time-dependent inhibition pattern:

This oxygen sensitivity profile demonstrates that:

• Brief oxygen exposure is relatively well-tolerated, with 80% activity retention after 5 minutes

• Prolonged exposure leads to substantial activity loss (70% reduction after 60 minutes)

• The inhibition is largely reversible, as >90% activity is recovered upon returning to anaerobic conditions

This reversibility suggests oxygen causes temporary inhibition rather than permanent denaturation of the enzyme. For P. profundum Hcp, which originates from an environment with variable oxygen levels, the oxygen sensitivity might be modulated compared to Hcps from strict anaerobes.

The practical implication for researchers is that hydroxylamine reductase assays should ideally be performed under anaerobic conditions to obtain optimal activity measurements.

What is the role of cyanide and carbon monoxide in modulating Hcp activity?

Cyanide (CN⁻) and carbon monoxide (CO) demonstrate complex concentration-dependent effects on Hcp hydroxylamine reductase activity, providing insights into the enzyme's catalytic mechanism:

Effects of CN⁻ present in assay solution:

• Concentrations >5 mM: Significant stimulation of NH₂OH reduction

• Concentrations <1 mM: Minimal stimulation

• CO saturation: Diminishes the stimulatory effect of CN⁻

Effects of pre-incubation with CN⁻:

• Concentrations <500 μM: Slight stimulation (maximum ~10% at 300 μM)

• Concentrations >500 μM: Significant inhibition

• CO co-treatment: Blocks both CN⁻-dependent stimulation and inhibition

• High CN⁻ inhibition (>500 μM): Irreversible, not recovered by CN⁻ removal or CO treatment

These patterns suggest that CN⁻ and CO likely interact with metal centers in Hcp, potentially modifying the redox properties or accessibility of the active site. The ability of CO to block CN⁻ effects suggests they may compete for the same binding sites.

For P. profundum Hcp research, these modulatory effects provide valuable tools for mechanistic studies and may have particular relevance given the potentially variable redox conditions in its deep-sea habitat.

What methods are used to express and purify recombinant P. profundum Hcp?

While the search results don't provide specific protocols for P. profundum Hcp, methodological approaches can be inferred from related studies. Based on the purification of P. profundum α-carbonic anhydrase (PprCA) and other bacterial Hcps, the following protocol can be outlined:

Expression System:

• Host: E. coli (commonly BL21(DE3) or similar expression strains)

• Vector: pET-based vectors containing the hcp gene with a histidine tag

• Induction: IPTG-inducible promoter system

Purification Protocol:

• Cell lysis under anaerobic conditions to preserve enzyme activity

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture His-tagged protein

• Buffer optimization containing reducing agents (DTT or β-mercaptoethanol) to maintain the iron-sulfur clusters

• Optional size exclusion chromatography for higher purity

• Storage under anaerobic conditions, preferably in buffer containing glycerol as a stabilizing agent

Activity Verification:

• Hydroxylamine reductase activity assays as described previously

• Spectroscopic characterization to verify the integrity of iron-sulfur clusters

Special Considerations for P. profundum Hcp:

• Expression at lower temperatures (15-20°C) to promote proper folding of a psychrophilic protein

• Inclusion of salt in purification buffers to maintain halotolerant enzyme stability

• Maintenance of anaerobic conditions throughout purification to preserve activity

What are the structural characteristics of P. profundum Hcp that contribute to its function?

While direct structural data for P. profundum Hcp is not available in the search results, we can infer key structural features based on related proteins and P. profundum's environmental adaptations:

Predicted Key Structural Elements:

• Iron-Sulfur Clusters: Likely contains the characteristic hybrid [4Fe-2S-2O] cluster that contributes to the distinctive spectroscopic properties of Hcps .

• Cysteine Residues and Disulfide Bridges: The search results note that "N-terminal cysteine residues (Cys 33 and Cys 186) [are] implicated in structural stability" of PprCA . Similar strategic positioning of cysteine residues may contribute to P. profundum Hcp stability.

• Salt Bridges and Surface Charge Distribution: As a halotolerant enzyme from a deep-sea bacterium, P. profundum Hcp likely features an optimized surface charge distribution that maintains protein solubility and function in high-salt environments, similar to PprCA which exhibits "salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions" .

• Cold Adaptation Features: Likely contains structural elements typical of psychrophilic enzymes, such as:

  • Reduced number of rigid proline residues

  • Increased surface hydrophilicity

  • Greater flexibility in catalytic regions

• Active Site Architecture: The hydroxylamine reductase activity suggests a specialized active site that can coordinate hydroxylamine while maintaining optimal electron transfer from physiological electron donors via Hcr.

These structural adaptations would enable P. profundum Hcp to function efficiently in the challenging deep-sea environment, characterized by high pressure, low temperature, and variable salinity.

How can site-directed mutagenesis be used to alter the catalytic properties of P. profundum Hcp?

While the search results don't directly address mutagenesis of P. profundum Hcp, strategic approaches can be designed based on insights from related enzymes:

Potential Mutagenesis Targets and Strategies:

• Metal Coordination Sites: The search results mention that "substitution of some key residues that bind the CODH C cluster" converted it from catalyzing CO oxidation to having "good hydroxylamine reductase activity" (H265V CODH mutant) . Similar modifications to metal-binding residues in P. profundum Hcp could alter substrate specificity or reaction rates.

• Cysteine Residues: Given the importance of cysteine residues in P. profundum enzyme stability , site-directed mutagenesis of specific cysteines could:

  • Assess their contribution to structural integrity

  • Determine their role in redox sensing

  • Identify residues involved in iron-sulfur cluster coordination

• pH-Responsive Residues: If P. profundum Hcp exhibits bimodal pH activity like PprCA , mutation of residues whose protonation states affect activity could:

  • Shift the pH optimum

  • Narrow or broaden the pH activity range

  • Identify key catalytic residues

• Halotolerance Engineering: Targeted mutations of surface residues to modify charge distribution could:

  • Enhance or reduce halotolerance

  • Investigate the structural basis of salt adaptation

  • Create variants optimized for different salt conditions

Experimental Design for Mutagenesis Studies:

Such studies would not only advance understanding of structure-function relationships in P. profundum Hcp but could potentially yield variants with enhanced stability or catalytic properties for biotechnological applications.

What is the evolutionary significance of hydroxylamine reductase activity in deep-sea bacteria like P. profundum?

The presence of hydroxylamine reductase activity in P. profundum likely represents an important evolutionary adaptation to its deep-sea niche:

• Adaptation to Nitrogen-Limited Environments: Deep-sea environments can be nutrient-limited, making efficient nitrogen metabolism advantageous. Hydroxylamine reductase activity enables the detoxification of hydroxylamine (a reactive intermediate in nitrite reduction) while simultaneously converting it to bioavailable ammonia , potentially allowing P. profundum to utilize a wider range of nitrogen sources.

• Response to Variable Redox Conditions: Deep-sea environments experience fluctuating oxygen concentrations, which can lead to incomplete denitrification and accumulation of reactive nitrogen species. The ability to detoxify these compounds through Hcp activity provides a selective advantage .

• Integration with Broader Metabolic Networks: The search results indicate that in some bacteria, Hcp regulation is linked to other metabolic pathways, such as sulfate reduction in Desulfovibrio species . This suggests evolutionary integration of nitrogen metabolism with other key cellular processes.

• Pressure and Cold Adaptation Co-Evolution: The hydroxylamine reductase activity in P. profundum has likely co-evolved with adaptations to high pressure and low temperature, resulting in an enzyme that functions optimally under deep-sea conditions.

• Horizontal Gene Transfer Considerations: The presence of Hcp across diverse bacterial lineages suggests possible horizontal gene transfer events in its evolutionary history. For P. profundum, acquisition of specialized hcp genes may have contributed to its ecological success in the deep sea.

From an evolutionary perspective, the maintenance of hydroxylamine reductase activity in P. profundum reflects the importance of nitrogen metabolism and detoxification mechanisms for survival in the challenging deep-sea environment.