Rapid and simple colorimetric method developed for screening of nitrogenase variants

Using this method, researchers found nitrogenase variants more resistant to oxygen and independent from ancillary chaperones.


Obtaining nitrogen-fixing plant crops is a reality that is getting closer and closer. However, the complexity of the nitrogen fixation process, together with the high sensitivity to oxygen of many of its components, makes it a challenge.

Biological nitrogen fixation is a natural process exclusive to some microorganisms such as bacteria or archaea. It consists of the transformation of atmospheric nitrogen (N2) into ammonium (NH3), which can be assimilated by other organisms, thanks to the action of an enzyme called nitrogenase. Since this capacity is totally absent in plants, a regular supply of nitrogen fertilizers is necessary to ensure good crop growth. Therefore, obtaining plants that produce the nitrogenase enzyme and that can manufacture their own nitrogen compounds could reduce serious environmental problems related to the excessive use of fertilizers. Although it seems a simple strategy, to obtain an active and functional nitrogenase requires the correct functioning of other proteins, apart from nitrogenase, that participate in nitrogen fixation, many of which are extremely sensitive to oxygen.

A clear example is the protein NifH, one of the components that make up the nitrogenase enzyme and that is responsible for sending electrons to its other component, NifDK, to transform N2 into NH3. NifH ceases to be active after a few seconds in the presence of oxygen and generally requires the action of another protein known as NifM for its correct conformation and function. Therefore, the study of different NifH proteins with better properties, such as independence from NifM or greater resistance to oxygen, could facilitate the development of plants with functional nitrogenases.

In the work developed by the Biochemistry of Nitrogen Fixation group of the CBGP, a fast and simple colorimetric assay has been adapted to measure the nitrogenase activity of several NifH proteins from different microorganisms. After an initial screening for stability and solubility in yeast cells, a eukaryotic model prior to the use of plants, the number of NifH candidates was reduced from 35 to 9. The nitrogenase activity of these 9 candidates was studied using a compound known as S2V with an intense blue color. This compound gradually loses its blue color and becomes transparent as NifH steals its electrons and yields them to NifDK. Only 3 NifH proteins showed activity in this assay, of which one was stable and active in the absence of NifM and another was more resistant to contact with oxygen than the NifH proteins characterized to date. As a result, these two variants could be good candidates for future expression in plants and be one step closer to achieving nitrogen-fixing plants.


Original Paper:

Payá-Tormo, L., Coroian, D., Martín-Muñoz, S., Badalyan, A., Green, R.T., Veldhuizen, M., Jiang, X., López-Torrejón, G., Balk, J., Seefeldt, L.C., Burén, S., Rubio, L.M. 2022. A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation. Scientific Reports 12, 10367. DOI: 10.1038/s41598-022-14453-x