Two Key Ferredoxins for Nitrogen Fixation Have Different Specificities and Biophysical Properties

Area of Science:

• Biochemistry and Molecular Biology
• Microbiology
• Structural Biology

Background:

• Ferredoxins are vital electron carriers for numerous biochemical reactions, including nitrogen fixation.
• Two specific ferredoxins, FdC and FdN, were previously identified as essential for iron nitrogenase-mediated nitrogen fixation in *R. capsulatus*.

Purpose of the Study:

• To investigate and delineate the key differences between FdC and FdN regarding their specificity, structure, and electronic properties.
• To elucidate the biophysical basis for ferredoxin specificity in nitrogen fixation.

Main Methods:

• In vivo complementation studies using gene deletion strains of *R. capsulatus*.
• Electron paramagnetic resonance (EPR) spectroscopy to analyze electronic features.
• X-ray crystallography for FdC structure determination and AlphaFold modeling for FdN structure prediction.

Main Results:

• Plasmid-based expression of *fdxN* restored diazotrophic growth and Fe-nitrogenase activity in both ∆fdxC and ∆fdxN strains.
• Plasmid-based expression of *fdxC* only complemented the ∆fdxC strain, indicating differential functional roles.
• EPR spectroscopy and structural analyses revealed significant differences in the electronic and structural properties of FdC and FdN, with novel structural features identified in FdC.

Conclusions:

• FdC and FdN possess distinct biophysical properties that dictate their specificity in the nitrogen fixation pathway.
• The study provides novel insights into the structural and electronic characteristics of ferredoxins essential for nitrogen (N₂) fixation.
• Understanding these differences is key to comprehending the intricate mechanisms of enzyme-catalyzed nitrogen fixation.