The "nitrogenase-protective" FeSII protein of Azotobacter vinelandii: overexpression, characterization, and crystallization

Abstract

The Azotobacter vinelandii FeSII protein confers conformational protection to nitrogenase by binding to the MoFe and Fe proteins under periods of oxidative stress to create an inactive but O2-stabilized tripartite complex. In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a homodimer and is expressed as a holoprotein with one [2Fe-2S]2+,+ cluster in each subunit. It is shown to be functional in reconstituting an O2-stable nitrogenase complex in vitro. Spectroscopic studies using the combination of UV-visible absorption, CD, and variable temperature MCD, EPR, and resonance Raman indicate that the [2Fe-2S]2+,+ cluster is coordinated exclusively by cysteine residues. The arrangement of coordinating cysteines in the primary sequence and the EPR properties of the [2Fe-2S]+ cluster (g = 2.04, 1.95, 1.88) are very similar to those of chloroplast ferredoxins. However, the variable-temperature MCD, resonance Raman, and redox properties (Em = -262 +/- 10 mV based on dye-mediated EPR redox titrations) are more characteristic of hydroxylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast-type ferredoxins, the vibrational properties of the [2Fe-2S]2+,+ cluster in the FeSII protein indicate that none of the cysteinyl Fe-S-C-C dihedral angles are close to 180 degrees and that the cluster is not exposed to solvent. Preliminary X-ray diffraction analysis indicates that the protein crystallizes in an orthorhombic space group with unit cell dimensions a = 135 A, b = 135 A, and c = 38 A and that there are at least two dimers per asymmetric unit.

Coordinated Expression of fdxD and Molybdenum Nitrogenase Genes Promotes Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Oxygen

“…capsulatus FdxD and A. vinelandii FeSII are plant-type ferredoxins coordinating 2Fe2S clusters by highly conserved cysteine residues (38,39). Unlike R. capsulatus FdxN and other ferredoxins, FdxD and FeSII are unable to serve as electron donors for nitrogenase due to their relatively high redox potentials (38)(39)(40). FeSII of A. vinelandii and G. diazotrophicus interact noncovalently with Mo-nitrogenase, thus mediating conformational protection against oxygen (10,11).…”

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

“…Conformational protection is due to the formation of a complex between the FeSII (Shethna) protein and nitrogenase under high intracellular oxygen concentrations. 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).…”

Role of the Azotobacter vinelandii Nitrogenase-Protective Shethna Protein in Preventing Oxygen-Mediated Cell Death

“…In this state, the nitrogenase component proteins are in an inactive but protected state capable of reconversion to the catalytically active state when the redox environment becomes favorable for nitrogenase function (11). The spectral and physical properties of FeSII (also known as the Shethna protein) of Azotobacter vinelandii has been well characterized, and the protein has been crystallized (8). It contains two [2Fe-2S] clusters that undergo oxidation-reduction, and it is presumed that this redox-active nature of the protein mediates its interactions with nitrogenase (8,13).…”

“…The spectral and physical properties of FeSII (also known as the Shethna protein) of Azotobacter vinelandii has been well characterized, and the protein has been crystallized (8). It contains two [2Fe-2S] clusters that undergo oxidation-reduction, and it is presumed that this redox-active nature of the protein mediates its interactions with nitrogenase (8,13). Specific amino acid residues important in the function, including in presumably recognizing the nitrogenase component proteins, have recently been proposed based on analysis of site-directed mutations within the FeSII protein (6).…”

“…A number of redox titration experiments performed using redox mediators and various O 2 levels have demonstrated that both the MoFe and Fe proteins of nitrogenase can exist in a number of reduced, oxidized, or intermediate redox states (18). It is likely that in the initial formation of the oxygen stable complex, the FeSII protein by virtue of its intermediate redox potential (8), can act as a redox mediator between the MoFe and Fe protein and O 2 , maintaining the components in a stable oxidized state. In the absence of the FeSII protein, however, oxidation of the nitrogenase components rapidly proceeds to an irreversibly oxidized, inactive state, and toxic O 2 -derived products may be generated as a consequence.…”