The FeSII protein of Azotobacter vinelandii has been proposed to mediate the 'conformational protection' of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII locus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesII locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is upregulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other sigma 70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCl (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen inactivation. Upon energy starvation, conditions in which 'respiratory protection' is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the 'iron-only' alternative nitrogenases exhibited similar growth rates irrespective of the presence or absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby 'respiratory protection' is the major physiological mechanism responsible for the protection of all three nitrogenases during energy-supplemented growth. Upon energy starvation, however, 'conformational protection', mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.