Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium.

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may fix nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Using a lacZ reporter with a fluorescent beta-galactoside substrate for in situ localization of gene expression, we have shown that the two clusters of nif genes are expressed independently. One nitrogenase functions only in heterocysts under either aerobic or anaerobic growth conditions, whereas the second nitrogenase functions only under anaerobic conditions in vegetative cells and heterocysts. Differences between the two nif clusters suggest that the nitrogenase that is expressed in heterocysts is developmentally regulated while the other is regulated by environmental factors.