The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen‐mediated inactivation of nitrogenase in vitro and in vivo

Abstract

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.

Evidence for Conformational Protection of Nitrogenase against Oxygen in Gluconacetobacter diazotrophicus by a Putative FeSII Protein

“…After 10 min, the activity had decreased to only 70% of that of the control extract incubated anaerobically with dithionite. This is in good agreement with results from similar experiments with Azotobacter extracts (14). Together, these results provide strong evidence for the protection of nitrogenase in G. diazotrophicus by a conformational mechanism involving a putative FeSII protein, which forms a complex with nitrogenase, rendering nitrogenase inactive but transiently protected from oxygen damage, similar to the situation in A. vinelandii.…”

“…Together, these results provide strong evidence for the protection of nitrogenase in G. diazotrophicus by a conformational mechanism involving a putative FeSII protein, which forms a complex with nitrogenase, rendering nitrogenase inactive but transiently protected from oxygen damage, similar to the situation in A. vinelandii. The specific signal leading to dissociation of the complex between nitrogenase and the FeSII protein has not been identified, although the redox status is believed to play a central role (14,21). The fact that cells that were switched from an atmosphere with 20% to one with 5% oxygen in N 2 with pyruvate did not regain activity indicates that pyruvate alone does not generate the signal for nitrogenase reactivation under these conditions, although dinitrogenase reductase is protected from degradation (Fig.…”

“…In this complex, nitrogenase is inactive but transiently protected from damage by oxygen (10,13,21). Additional studies have shown that the function of the FeSII protein is of major importance for cell viability under carbon and/or energy limitation but only when cells are grown diazotrophically (12,14).…”

Cytochrome c terminal oxidase pathways of Azotobacter vinelandii: analysis of cytochrome c4 and c5 mutants and up-regulation of cytochrome c-dependent pathways with N2 fixation

“…PCR was performed with 10 g of strain CA genomic DNA and 160 pmol of each degenerate primer under conditions described previously (27). The PCR fragment was radiolabeled as described elsewhere (31) and used to screen a previously constructed A. vinelandii genomic EMBL3 library (24). After a secondary screen of positive plaques, single phage plaques were selected to produce higher-titer stocks, and phage DNA was analyzed by enzymatic restriction.…”

Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase

“…It has been proposed (3) that Azotobacter utilizes two distinct strategies to reconcile these interdependent metabolic requirements. The first strategy, termed "conformational protection" (3), involves the formation of an O 2 -stable tripartite complex between a [2Fe-2S] protein and the nitrogenase subunits (23,28). In the second strategy, termed "respiratory protection" (3), vigorous oxygen consumption by the Azotobacter electron transport chain results in the maintenance of the low intracellular oxygen levels that are essential for nitrogen fixation.…”