IscA, an alternate scaffold for Fe-S cluster biosynthesis

An IscA homologue within the nif regulon of Azotobacter vinelandii, designa ted (Nif)IscA, was expressed in Escherichia coli and purified to homogeneit y. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that c ontained no metal centers or other prosthetic groups in its as-isolated for m. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorpti on, Mossbauer, resonance Raman, variable-temperature magnetic circular dich roism, and EPR spectroscopies, Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environ ment, the low-binding affinity argues against a specific role as a metalloc haperone for the delivery of ferrous ion to other Fe-S cluster assembly pro teins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe -S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly proces s was monitored temporally using UV-vis absorption and Mossbauer spectrosco py, and the intermediate [2Fe-2S](2+)-containing species was additionally c haracterized by resonance Raman spectroscopy. The Mossbauer and resonance R aman properties of the [2Fe-2S](2+) center are consistent with complete cys teinyl ligation. The presence of three conserved cysteine residues in all I scA proteins and the observed cluster stoichiometry of approximately one [2 Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster type s are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The abil ity of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-d irected cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.