Gene Cloning , Purification , and Characterization of Two Cy Homologs Driving Iron-Sulfur Cluster Formation

isms. Cysteine desulfurase, originally identified in a nitrogen-fixing bacterium, Azotobacter vinelandii, and named NifS, is essential for the production of a holo-form of nitrogenase in vivo. The gene coding for NifS (ntfS) is in the nif gene cluster, which contains genes encoding nitrogenase and other proteins involved in nitrogen fixation. NifS was shown to facilitate in vitro reconstitution of iron-sulfur clusters of nitrogenase from A. vinelandii4) as well as dihydroxy-acid dehydratase,S) SoxR,6) and FNR'} from Escherichia coli. The enzyme catalyzes the degradation of L-cysteine to produce L-alanine and elemental sulfur, which is used for the iron-sulfur cluster assembly. IscS, another enzyme from A. vinelandii, has a sequence similar to NifS, catalyzes the same reaction as NifS, and is involved in the ironsulfur cluster formation. It is believed that, in vivo, IscS plays a general role in iron-sulfur cluster assembly, whereas NifS is specialized in the maturation of nitrogenase. 6enes with sequence similarity to n(rs and iscS have been found in a variety of organisms, suggesting that NifS homologs play a role in the ironsulfur cluster formation in a wide range of organisms. niLfU in the nifgene cluster is another essential gene for the full activation of nitrogenase, It was proposed recently that NifU from A. vinelandii cooperates with NifS in the maturation of iron-sulfur proteins by providing an intermediate iron-sulfur assembly site: NifU and NifS interact with each other, and