Recombinant Nitrogen fixation protein nifU (nifU)

What is NifU and what role does it play in biological nitrogen fixation?

NifU serves as a molecular scaffold for the assembly of transient iron-sulfur clusters that are subsequently transferred to target proteins in the nitrogen fixation pathway. Research demonstrates that NifU transfers [Fe4-S4] clusters to three major sets of iron-sulfur proteins involved in biological nitrogen fixation: NifH (the Fe protein of nitrogenase), NifB, and NifQ . This transfer is essential for the maturation and activation of these proteins, making NifU a central player in biological nitrogen fixation.

What is the domain structure of NifU protein?

NifU from Azotobacter vinelandii exemplifies domain evolution by combining three distinct domains within a single polypeptide:

• N-terminal IscU scaffold motif - Has the dominant function in nitrogenase-specific iron-sulfur cluster formation

• Central ferredoxin fold

• C-terminal NfuA-like domain

Both the N-terminal and C-terminal domains contain conserved cysteine residues that serve as potential iron-sulfur cluster assembly sites. Genetic experiments involving amino acid substitutions within these domains indicate that both can separately participate in nitrogenase-specific iron-sulfur cluster formation, although the N-terminal domain typically plays the dominant role .

How does NifU interact with other proteins in the nitrogen fixation pathway?

NifU engages in specific interactions with several key proteins:

• NifS: Works in conjunction with NifU as a pyridoxal phosphate-dependent cysteine desulfurase, providing the sulfur needed for cluster assembly by catalyzing the removal of sulfur from L-cysteine .

• NifQ: Directly interacts with apo-NifQ (NifQ without its iron-sulfur cluster). This interaction is only effective when NifQ is unoccupied by its iron-sulfur cluster. The iron content of apo-NifQ increases after incubation with as-isolated NifU .

• Fe protein (NifH): NifU facilitates the maturation of nitrogenase Fe protein by enabling the assembly and transfer of iron-sulfur clusters, as demonstrated in experiments showing NifU-mediated activation of apo-Fe protein .

What are the common methods for recombinant expression of NifU?

Several expression systems have been developed for recombinant NifU production:

• Bacterial Expression Systems: High-level heterologous expression of A. vinelandii NifU in E. coli using plasmid constructs allows for co-expression with NifS, which is often necessary for proper function .

• Plant Expression Systems: For plant biotechnology applications, synthetic gene design with codon optimization and appropriate promoters (such as p35S) has been employed to enhance NifU expression .

• Chloroplast Targeting Approaches: For expression in plant chloroplasts, NifU has been fused with chloroplast transit peptides (CTPs) to direct the protein to the chloroplast compartment .

Key considerations for successful expression include:

• Maintaining anaerobic conditions to prevent oxidative damage

• Co-expressing NifU with NifS for proper function

• Using appropriate tags (e.g., His-tags) for purification

• Optimizing codons for the host organism

How can NifU activity be measured in vitro?

Researchers employ several complementary methods to assess NifU activity:

• Iron-Sulfur Cluster Transfer Assay: Directly measures transfer of iron-sulfur clusters from NifU to target proteins:

  • Prepare apo-Fe protein by removing its iron-sulfur cluster through chelation

  • Incubate NifU with NifS, Fe²⁺, and L-cysteine to generate transient iron-sulfur clusters

  • Mix NifU containing transient clusters with apo-Fe protein

  • Measure Fe protein activity through acetylene reduction assay

• Iron Content Analysis: Quantifies iron transfer using:

  • Inductively coupled plasma-mass spectrometry (ICP-MS)

  • Colorimetric assays with iron chelators

• Spectroscopic Methods:

  • UV-visible absorption spectroscopy

  • Electron paramagnetic resonance (EPR) spectroscopy

  • Mössbauer spectroscopy

What protein purification techniques are most effective for NifU?

Based on published research, successful purification of NifU typically involves:

• Affinity Chromatography: Using tagged versions of NifU (e.g., His-tagged NifU) with metal affinity columns like Ni-NTA . This approach is particularly valuable for studying protein-protein interactions.

• Anaerobic Purification: Due to oxygen sensitivity of iron-sulfur clusters, purification under strictly anaerobic conditions is essential to maintain cluster integrity and protein activity .

• Size Exclusion Chromatography: Often used as a final polishing step to ensure high purity and separate different oligomeric states of NifU.

• Ion Exchange Chromatography: Separates NifU based on charge properties, particularly useful for differentiating between apo-NifU and holo-NifU forms.

A typical purification protocol includes:

• Cell lysis under anaerobic conditions

• Initial capture using affinity chromatography

• Further purification using ion exchange and/or size exclusion chromatography

• Concentration and storage under anaerobic conditions

How does NifU transfer iron-sulfur clusters to target proteins?

The mechanism of iron-sulfur cluster transfer from NifU to target proteins involves direct protein-protein interactions rather than simple dissociation and reassociation of iron:

• Direct Protein-Protein Transfer: Experiments with apo-NifQ and NifU separated by a membrane that only allowed for iron diffusion showed no iron transfer to apo-NifQ, demonstrating that direct protein contact is required .

• Domain-Specific Transfer: Both N-terminal and C-terminal domains of NifU can participate in iron-sulfur cluster formation and transfer. Mutation studies show that inactivation of the N-terminal scaffold diminishes but doesn't eliminate in vitro activation of apo-Fe protein, while inactivation of both domains completely eliminates this capacity .

• Concentration-Dependent Activation: The activation of apo-Fe protein by NifU follows a concentration-dependent pattern, with maximum activity at approximately equimolar concentrations of NifU and apo-Fe protein .

What is the relationship between NifU and NifQ in molybdenum trafficking?

The NifU-NifQ relationship represents a critical junction where iron and molybdenum metabolism coordinate in biological nitrogen fixation:

• Iron-Sulfur Cluster Transfer: NifU transfers an iron-sulfur cluster to apo-NifQ, which is necessary for NifQ to form a [MoFe3S4] group. This molybdenum-containing cluster then serves as a specific donor during nitrogenase cofactor biosynthesis .

• Protein-Protein Interaction: Protein-protein interaction studies have demonstrated that apo-NifQ and as-isolated NifU interact directly. This interaction is only effective when NifQ lacks its iron-sulfur cluster .

• Iron Transfer Mechanism: The apo-NifQ iron content increases after incubation with as-isolated NifU, reaching similar levels to holo-NifQ. This transfer depends on direct protein-protein interaction and cannot occur through simple iron diffusion .

• Co-regulation System: The relationship suggests a regulatory mechanism where sufficient iron must be allocated for NifU before molybdenum can be used for NifQ, ensuring proper coordination of both elements for efficient nitrogen fixation .

How do mutations in different domains of NifU affect its function?

Research on NifU mutations has revealed important insights into domain functionality:

• N-terminal Domain Mutations:

  • Substitution of any or all three conserved cysteines (Cys35, Cys62, and Cys106) with alanine results in similar diazotrophic growth phenotypes, indicating that nitrogenase maturation doesn't absolutely require a functional N-terminal domain .

  • In vitro studies show these mutations diminish but don't eliminate capacity for activation of apo-Fe protein .

• C-terminal Domain Mutations:

  • The C-terminal domain contains conserved cysteines that can also serve as a scaffold for iron-sulfur cluster assembly.

• Combined Domain Mutations:

  • When both N-terminal and C-terminal potential assembly sites are inactivated, in vitro apo-Fe protein activation is completely eliminated .

These findings indicate NifU possesses functional redundancy, with both domains capable of participating in iron-sulfur cluster assembly and transfer, though the N-terminal domain typically plays the dominant role.

What challenges exist in expressing functional NifU in non-native hosts?

Expressing functional NifU in non-native hosts presents several significant challenges:

• Oxygen Sensitivity: NifU and its iron-sulfur clusters are highly sensitive to oxygen, making expression in aerobic environments particularly challenging, especially in plant systems that generate oxygen during photosynthesis .

• Co-expression Requirements: Functional NifU often requires co-expression with other proteins, particularly NifS, adding complexity to heterologous expression systems .

• Cellular Compartmentalization: In plant systems, targeting NifU to the appropriate subcellular compartment (chloroplasts or mitochondria) is crucial for functionality .

• Post-translational Modifications: Proper folding and potential post-translational modifications may vary between host organisms.

• Codon Usage Differences: Differences in codon usage between native organism and host can significantly affect expression levels .

How is NifU being used in engineering nitrogen fixation in plants?

NifU plays a critical role in engineering nitrogen fixation in plants through several approaches:

• Chloroplast Targeting: Research has demonstrated successful targeting of NifU to plant chloroplasts using chloroplast transit peptides. The processed NifU proteins show correct import and cleavage, generating chloroplast-recombinant Nif proteins .

• Co-expression Strategy: NifU is being co-expressed with other nitrogenase components, including NifH, NifB, and NifQ, to establish a functional nitrogen fixation pathway in plants .

• Synthetic Gene Design: Synthetic gene design approaches have optimized NifU expression in plants, resulting in significantly increased accumulation of Nif proteins compared to native gene sequences .

• Functional Validation: Co-expression with NifU has been shown to be essential for NifH activity when purified from plant chloroplasts, as well as for NifB obtained from yeast mitochondria .

Research indicates that similar co-expression with NifU would be needed for a functional NifQ-mediated molybdenum delivery pathway to nitrogenase in plants .

What optimization strategies have been effective for improving NifU function in heterologous systems?

Several optimization strategies have proven effective for improving NifU function:

• Synthetic Gene Design: The use of synthetically optimized genes has shown dramatically increased accumulation of Nif proteins. In one study, NifH accumulated 170 times more when a synthetic gene was used for expression .

• Codon Optimization: Adjusting codon usage to match the host organism has significantly improved expression levels .

• Optimized Chloroplast Transit Peptides: Selection of appropriate chloroplast transit peptides has improved targeting and processing of NifU in plant chloroplasts .

• Controlled Expression Timing: Following the accumulation of NifU through a time course has optimized protein yields. One study found that NifH levels steadily increase until the end of the dark period of the third day post-agroinfiltration .

• Co-expression with Partner Proteins: Co-expression of NifU with functional partners, particularly NifS, improves assembly and transfer of iron-sulfur clusters .

Why might recombinant NifU be inactive in vitro?

Several factors can contribute to inactivity of recombinant NifU:

• Oxidative Damage: Exposure to oxygen can damage iron-sulfur clusters or prevent their proper assembly. Ensuring strictly anaerobic conditions during purification and assays is critical .

• Improper Folding: Misfolding due to expression conditions or host factors can lead to inactive protein.

• Absence of Co-factors: NifU requires proper co-factors, particularly iron, for functionality.

• Missing Partner Proteins: NifU typically functions with NifS, which provides sulfur for iron-sulfur cluster assembly. Absence of NifS can lead to inactive NifU .

• Mutations or Truncations: Unintended mutations or truncations can affect activity. Both N-terminal and C-terminal domains contain important cysteine residues necessary for iron-sulfur cluster assembly and transfer .

• Improper Buffer Conditions: Buffer composition, pH, and ionic strength can significantly affect NifU stability and activity.

How can stability of NifU and its iron-sulfur clusters be maintained during experiments?

Maintaining NifU stability requires careful attention to several factors:

• Anaerobic Conditions: All procedures involving NifU should be conducted under strictly anaerobic conditions to prevent oxidative damage to iron-sulfur clusters .

• Reducing Agents: Including appropriate reducing agents (DTT, β-mercaptoethanol, or sodium dithionite) in buffers helps maintain the reduced state of iron-sulfur clusters .

• Buffer Optimization: Buffer systems that include stabilizing agents such as glycerol can significantly enhance NifU stability.

• Temperature Control: Performing experiments at lower temperatures (e.g., 4°C) can reduce thermal denaturation.

• Protein Concentration Management: Working with appropriate protein concentrations prevents aggregation or precipitation.

• Appropriate Storage: Storage in liquid nitrogen or at -80°C under anaerobic conditions helps maintain long-term stability.

• Iron and Sulfur Sources: For experiments involving iron-sulfur cluster assembly, ensuring adequate supplies of Fe²⁺ and sulfur (typically provided by NifS-mediated cysteine desulfuration) is critical .

What are common pitfalls in NifU-related experimental design?

Researchers should be aware of several common pitfalls:

• Insufficient Anaerobic Precautions: Failure to maintain strictly anaerobic conditions throughout all experimental stages can lead to oxidative damage and inactivation .

• Overlooking Protein-Protein Interactions: NifU functions through direct protein-protein interactions with target proteins. Experimental designs that rely on simple diffusion rather than direct protein contact may fail to capture the true biological mechanism .

• Inadequate Controls: When studying iron transfer from NifU to NifQ, controls with membrane separation demonstrated that direct protein-protein interaction was necessary .

• Neglecting Domain-Specific Functions: Experiments focusing on only one domain may miss important functional aspects of the protein .

• Time-Dependent Transfer: Iron-sulfur cluster transfer can be time-dependent. Incubation times of 5 minutes versus 120 minutes can yield significantly different results in iron transfer from NifU to NifQ .

• Concentration Dependencies: The activation of apo-Fe protein by NifU shows a concentration-dependent pattern, with maximum activity at approximately equimolar concentrations. Non-optimal ratios may underestimate NifU activity .