Nitrogenase is the key enzyme responsible from nitrogen fixation. However, this enzyme is oxygen-sensitive and its activity is irreversibly inhibited by oxygen. It is said that a few bacteria have nitrogenases that can still fix N2 in the presence of O2.
Dear Daniel Deng,
Yes the nitrogenase from aerobic microorganisms can tolerate oxygen. It is the Fe Protein subunit which is oxygen sensitive. The O2 response is mediated via nif L gene product in K.pneummoniae and fix L, J and K products in Rhizibia. So you can explore considering these options for engineering nitrogenase.
Rekha Gadre
Dear Daniel,
There is, to my knowledge, a single case documented: that of the nitrogenase from Azotobacter chroococcum. Itw as documented in a paper published in Biochem Journal 1979 (wow almost forty years ago) 181(3): 569-575
Here is the abstract:
In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe--2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO--Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of 'conformational protection' in this organism.
From what I read in this paper, the tolerance to O2 is communicated to the nitrogenase through the binding of a small 14-kDa iron-sulfur protein.
An idea could be to isolate the gene of this iron-sulfur proteina nd add it to several oxygen-sensitive nitrogenases and look if they becoem more oxygen-tolerant?
Best regards,
Pr Philippe Urban
Dear Daniel Deng,
While navigating search for nitrogen fixation and oxygen sensitivity, I came across with two important hits. Of course, one was yours about engineering oxygen-tolerant Nitrogenase, and second, of which I think, I am sure you may be aware about NSF funded research project on "Oxygen-Tolerant Nitrogenase". I am sure, if these investigators are successful in re-discovering oxygen-insensitive isolate Streptomyces thermoautotrophicus strain, we all will be lucky to have oxygen-tolerant Nitrogenase. Meanwhile, I think, you may think to make use of info shared by earlier researchers.
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