A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Area of Science:

• Biochemistry
• Molecular Biology
• Agricultural Science

Background:

• Biological nitrogen fixation (BNF) converts atmospheric nitrogen (N2) to ammonia (NH3) using oxygen-sensitive nitrogenase enzymes in prokaryotes.
• Engineering crops for direct BNF could reduce synthetic fertilizer reliance and boost agricultural sustainability.
• Current acetylene reduction assay (ARA) for nitrogenase activity is not suitable for high-throughput screening or automation.

Purpose of the Study:

• To develop a microplate-based assay for high-throughput screening of nitrogenase components.
• To identify NifH proteins with desirable traits for engineering nitrogen fixation in plants.
• To assess NifH proteins for NifM independency and oxygen (O2) resistance.

Main Methods:

• Developed and validated a reduced sulfonated viologen derivative (S2Vred) assay for measuring nitrogenase activity using a microplate reader.
• Screened a library of NifH proteins targeted to yeast mitochondria.
• Evaluated NifH proteins for NifM dependency, O2 tolerance, solubility, and [Fe-S] cluster occupancy.

Main Results:

• The S2Vred assay successfully replaced the ARA for simultaneous analysis of isolated nitrogenase proteins.
• Identified NifH from *Dehalococcoides ethenogenes* as NifM-independent, requiring fewer genes for transfer to eukaryotes.
• Identified NifH from *Geobacter sulfurreducens* as exhibiting enhanced O2 resistance, crucial for stability in eukaryotic cells.

Conclusions:

• The S2Vred assay is a viable, high-throughput alternative to ARA for nitrogenase activity screening.
• Specific NifH proteins possess advantageous biochemical properties (NifM independency, O2 tolerance) for engineering BNF in plants.
• Focusing on identifying such naturally occurring nitrogenase components is key for developing self-fertilizing crops.