THE NITROGENASE-PROTECTIVE FESII PROTEIN OF AZOTOBACTER-VINELANDII - OVEREXPRESSION, CHARACTERIZATION, AND CRYSTALLIZATION

The Azotobacter vinelandii FeSII protein confers conformational protec tion to nitrogenase by binding to the MoFe and Fe proteins under perio ds of oxidative stress to create an inactive but O-2-stabilized tripar tite complex, In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to ho mogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a hom odimer and is expressed as a holoprotein with one [2Fe-2S](2+.+) clust er in each subunit. It is shown to be functional in reconstituting an O-2-stable nitrogenase complex in vitro. Spectroscopic studies using t he 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 c oordinating cysteines in the primary sequence and the EPR properties o f the [2Fe-2S](+) cluster (g = 2.04, 1.95, 1.88) are very similar to t hose of chloroplast ferredoxins. However, the variable-temperature MCD , resonance Raman, and redox properties (E(m) = -262 +/- 10 mV based o n dye-mediated EPR redox titrations) are more characteristic of hydrox ylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast -type ferredoxins, the vibrational properties of the [2Fe-2S](2+.+) cl uster 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 Angstrom, b = 135 Angstrom, and c = 38 Angstr om and that there are at least two dimers per asymmetric unit.