Characterization of an Iron−Sulfur Cluster Assembly Protein (ISU1) from Schizosaccharomyces pombe

Abstract

Genetic studies of bacteria and eukaryotes have led to identification of several gene products that are involved in the biosynthesis of protein-bound iron-sulfur clusters. One of these proteins, ISU, is homologous to the N-terminus of bacterial NifU. The mature forms of His-tagged wild-type and D37A Schizosaccharomyces pombe ISU1 were cloned and overexpressed as inclusion bodies in Escherichia coli. The recombinant D37A protein was purified under denaturing conditions and subsequently reconstituted in vitro. By use of a 5-fold excess of iron and sulfide the reconstituted product was found to be red-brown in color, forming a homodimer of 17 kDa per subunit with approximately two iron atoms per monomer determined by protein and iron quantitation. UV-vis absorption and Mössbauer spectroscopies (delta = 0.29 +/- 0.05 mm/s; DeltaE(Q) = 0.59 +/- 0.05 mm/s) were used to characterize D37A ISU1 and show the presence of [2Fe-2S](2+) clusters in each subunit. Formation of the holo form of wild-type ISU1 was significantly less efficient using the same reconstitution conditions and is consistent with prior observations that the D37A substitution can stabilize protein-bound clusters. Relative to the human homologue, the yeast ISU is significantly less soluble at ambient temperatures. In both cases the native ISU1 is more sensitive to proton-mediated degradation relative to the D37A derivative. The lability of this family of proteins relative to [2Fe-2S] bearing ferredoxins most likely is of functional relevance for cluster transfer chemistry. Mössbauer parameters obtained for wild-type ISU1 (delta = 0.31 +/- 0.05 mm/s; DeltaE(Q) = 0.64 +/- 0.05 mm/s) were similar to those obtained for the D37A derivative. Cluster transfer from ISU1 to apo Fd is demonstrated: the first example of transfer from an ISU-type protein. A lower limit for k(2) of 80 M(-1) min(-1) was established for WT cluster transfer and a value of 18 M(-1) min(-1) for the D37A derivative. Finally, we have demonstrated through cross-linking studies that ferredoxin, an electron-transport protein, forms a complex with ISU1 in both apo and holo states. Cross-linking of holo ISU1 with holo Fd is consistent with a role for redox chemistry in cluster assembly and may mimic the intramolecular complex already defined in NifU.

Iron Sulfur Cluster Biosynthesis

“…In agreement with Dean and colleagues, 27 we found that IscU coordinates one reductively labile [2Fe-2S] 2+ cluster per monomeric subunit that is stabilized by substitution of a highly conserved aspartate (D37 for Hs ISU and Sp ISU, and D40 for Tm IscU), as judged by Mössbauer, EXAFS, EPR, and UV-vis spectroscopies. 44,45 Our studies of cluster stability and cluster transfer suggests that this highly conserved acidic residue maintains a solvent-accessible channel to the cluster. 46 IscU proteins are generally dimers, 47,48 with the exception of human ISU, which is a monomer.…”

“…Since Fe-S clusters typically require four cysteine ligands, it seemed plausible that the three cysteinecontaining IscUs coordinate one interfacial Fe-S cluster between two monomeric subunits while four cysteinecontaining IscUs bind one cluster per monomer. However, site-directed mutagenesis studies, 44 iron-to-protein ratios, 44,45 and correlations of cysteine content to oligomeric state 45 indicate that one Fe-S cluster coordinates to one monomeric subunit of IscU regardless of cysteine content. Also, comparison of UV-vis absorption, near-UV-vis CD, and Mössbauer spectra are consistent with a similar Fe-S cluster environment for all IscUs studied thus far.…”

“…Data from in vitro biochemical and spectroscopic studies 27,44,45,49 coupled with results from in vivo experiments 33,39,40,50 strongly suggested that IscU is a biological scaffold for Fe-S cluster assembly. Further support for such a role came from far-UV CD 26 and NMR 48 studies, which showed essentially identical secondary structural content and similar tertiary structural and dynamic properties for apo and holo T. maritima IscU, i.e., unlike other low molecular weight metalloproteins, IscU retains its tertiary structure in the apo state that is preformed for Fe-S cluster coordination.…”

Iron-Sulfur Cluster Biosynthesis

“…Coordination of the [2Fe-2S] 2ϩ cluster to IscU is stabilized by substitution of a highly conserved Asp with Ala at position 40 (Thermotoga maritima numbering) (1,3,5,6). Other characterized proteins within this system include IscS (which delivers sulfur to IscU) (1), IscA (7-10), a [2Fe-2S] ferredoxin (11,12), IscR (13), and chaperones Hsc66 and Hsc20 (14).…”

“…In recent reports, we characterized the factors influencing the stability of IscU-bound clusters (12) and the reactions of IscU in cluster transfer to a target protein, apoferredoxin (11). We also addressed the mechanism of assembly of IscU-bound clusters (15) and identified a natural iron delivery protein (16).…”