NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

The Nifs and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. Nifs is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained with in the nitrogenase component proteins, NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+,+) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with Nifs and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of Nifs. Approximately one transient [2Fe-2S] cluster is assembled per homodimer, The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU, Because homologs to both NifU and Nifs, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-s cluster-containing proteins.