Nitrogenases are the only enzymes able to "fix" atmospheric N2 into bioavailable NH3 and hence are essential for sustaining biological life. Nitrogenase catalysis is dependent on an ample supply of both ATP and electrons, the latter provided by low-potential redox proteins, often ferredoxins (Fds). Regarding this electron transport to nitrogenase proteins, many open questions remain; specifically electron transport to the iron (Fe-) nitrogenase had hardly been investigated. Our goal was to identify which Fds are important for nitrogen fixation by the Fe-nitrogenase and try to elucidate their functions.
Highlighted in our recent study, we identified two essential Fds for nitrogen fixation by the Fe-nitrogenase in Rhodobacter capsulatus (R. capsulatus)[1]. Genetic deletions of the Fd genes (fdx) in R. capsulatus were constructed, in a molybdenum nitrogenase deletion strain, and characterised via diazotrophic growth rates and in vivo nitrogenase activity. Deletion of two fdx genes, fdxC and fdxN, slowed diazotrophic growth and lowered Fe-nitrogenase activities. The ∆fdxN and ∆fdxC deletion strains were further studied via whole cell proteomics and plasmid complementation experiments. Proteomics revealed both strains had an upregulation of proteins essential for electron delivery to nitrogenases, indicating an interruption in electron transport to the Fe-nitrogenase. Plasmid complementation patterns differed greatly for ∆fdxN and ∆fdxC, revealing the two proteins were likely not redundant in function, but instead performed distinct roles.
To further explore the functions of FdN and FdC, our current research has focused on studying FdN and FdC in vitro. Specifically, FdN and FdC have been characterised spectroscopically, structurally and biochemically. Both Fds have been purified with redox-active FeS centres, shown by metal content analysis and UV-Vis spectroscopy, under reductive and oxidative conditions. Additionally, we have obtained an atomic resolution structure of one of the Fds, which has provided new details about the FeS cluster coordination, protein architecture and hydrogen bonding network. Overall, our findings have provided two key protein targets for the further study and bioengineering of nitrogen fixation systems, specifically those focussed on increasing the electron flux to nitrogenases for increased product formation.
1 Addison, H., …, Rebelein J. (2024) Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus. mBio. 15, e03314-03323