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.