Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. Owing to their remarkable structural plasticity and versatile chemical/electronic features [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Three different types of [Fe-S] cluster biosynthetic systems have been discovered, and all of them are mechanistically unified by the requirement for a cysteine desulfurase and the participation of an [Fe-S] cluster scaffolding protein. Important mechanistic questions related to [Fe-S] cluster biosynthesis involve the molecular details of how [Fe-S] clusters are assembled on scaffold proteins, how [Fe-S] clusters are transferred from scaffolds to target proteins, how various accessory proteins participate in [Fe-S] protein maturation, and how the biosynthetic process is regulated.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. 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 absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. 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 process was monitored temporally using UV-vis absorption and Mössbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types 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 ability 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-directed 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.

The process of Fe-S cluster biogenesis is highly conserved in nature and centers around two key proteins, termed IscS and IscU in prokaryotes, which are found in almost all organisms. 1 IscS is a homodimeric, pyridoxal phosphate-dependent L-cysteine desulfurase that catalyzes the conversion of cysteine to alanine and elemental sulfur via the formation of a persulfide intermediate on a conserved cysteine residue. 2 The ubiquitous role of IscSlike enzymes in sulfur trafficking, in general, 3 and Fe-S cluster biosynthesis, in particular, 4 is now firmly established. IscU is a homodimeric protein that forms a 1:1 complex with IscS, 5 and gene knockout studies in yeast 6 and bacteria 4d have demonstrated a crucial role for IscU in general Fe-S cluster biosynthesis. Moreover, IscU contains three conserved cysteines and has been shown to provide a scaffold for IscS-directed sequential assembly of [Fe 2 S 2 ] 2+ and [Fe 4 S 4 ] 2+ clusters. 7 These clusters are likely to be inserted, intact, into apo Fe-S proteins in a process that has yet to be fully characterized. In this communication, we address the mechanism of IscS-directed Fe-S cluster assembly on the IscU scaffold. Mass spectrometry has been used to demonstrate direct transfer of sulfane sulfur, S 0 , from the cysteine persulfide on IscS to the cysteine residues on IscU. The available evidence indicates that this reaction constitutes the first step in Fe-S cluster biosynthesis on an IscU scaffold.A comparison of the mass spectra of Azotobacter Vinelandii IscU obtained under a variety of reaction conditions is shown in Figure 1. Reaction mixtures were purified by reverse-phase HPLC, and discrete fractions were introduced by an electrospray ionization (ESI) source for analysis using a single quadrupole mass spectometer (see Supporting Information for experimental details). 8 The conditions used for repurification are denaturing, and the observed charged-state distributions, centered around m/z ≈ 1000 amu, are characteristic of highly denatured proteins. Hence, all observed molecular ions must be covalently associated.The mass spectrum of IscU after DTT treatment ( Figure 1A) comprises the monomer molecular ion peak at 13 744 Da (theoretical mass based on primary sequence, 13 875 Da, minus 131 Da, corresponding to cleavage of the N-terminal methionine), and features at 13 774 and 13 805 Da. Since the two higher mass components occur at +30 and +61 Da, respectively, and are dramatically decreased in intensity on addition of excess potassium cyanide ( Figure 1E), they are attributed to the addition of one and two sulfur atoms, respectively, in DTT-inaccessible polysulfides or persulfides. Direct evidence for multiple sulfur transfers from IscS to IscU is provided by the mass spectrum of IscU in a reaction mixture involving stoichiometric IscS and IscU with an excess of the substrate, cysteine ( Figure 1B). The major peaks at 13 805 and 13 836 Da correspond to the addition of two and three sulfur atoms, respectively, and peaks corresponding to the addition of one sulfu...