The Azotobacter vinelandii nifS gene product has been used with selenocysteine to reconstitute Klebsiella pneumoniae nitrogenase Fe protein. Chemical analysis and extended X-ray absorption fine structure (EXAFS) spectroscopy show that the 4Fe4S cluster present in the native protein is replaced by a 4Fe4Se cluster. As well, EXAFS spectroscopy shows that the bond lengths to the cysteine thiolate ligands shrink by 0.05 Å (from 2.28 to 2.23 Å) upon reduction, whereas the Fe-Fe distance is essentially unchanged. Thus, the core of the 4Fe4Se cluster remains essentially static on reduction, whilst the external cysteine thiolate ligands are pulled in towards the cluster. Compared with native (S)-Fe protein, the (Se)-Fe protein has a 20-fold increased rate of MgATP-induced Fe chelation, a sixfold decreased specific activity for acetylene reduction, a fivefold decreased rate of MgATP-dependent electron transfer from (Se)-Fe protein to MoFe protein, and a fourfold increase in the ATP to 2e- ratio. The high ATP to 2e- ratio and decreased specific activity are consistent with a lower rate of dissociation of oxidized (Se)-Fe protein from reduced MoFe protein. Thus, the relatively small adjustments in the Fe protein structure necessary to accommodate the 4Fe4Se cluster are transmitted both to adjacent residues that dock at the surface of the MoFe protein and to the ATP hydrolysis sites located approximately 19 Å away.