ISCU

Abstract: Fe-S cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into its target protein by an unknown mechanism. In Escherichia coli, genes in the ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 cluster (the isc gene cluster) should be involved in the assembly of the Fe-S cluster since its coexpression with the reporter ferredoxin (Fd) dramatically increases the production of holoFd [Nakamura, M., Saeki, K., and Takahashi, Y. (1999) J. Biochem. 126, 10-18]. In this study we addressed the functional roles of the proteins encoded by the isc gene cluster with respect to the assembly of Fe-S clusters in four reporter Fds. Plasmids were constructed in which eight ORFs in the isc gene cluster were individually inactivated either by truncating the coding region or by introducing an oligonucleotide linker containing stop codons. By coexpressing these plasmids with reporter Fds, we show the iscS, iscA, hscA, and fdx genes to be required for the assembly of the Fe-S clusters. When these genes were absent from the coexpression plasmid, no overproduction was achieved in any reporter Fds examined. The inactivation of ORF2 and hscB had a partial but appreciable effect on the production of some Fds. Deletion of ORF1 produced no difference from the coexpression with the intact isc gene cluster. We also examined coexpression using the fdx gene in the isc gene cluster as a reporter Fd and identified iscS, hscB, hscA, and ORF3 as being involved in the assembly of the [2Fe-2S] cluster in this protein. We propose a model in which the fdx gene product functions as an intermediate site for Fe-S cluster assembly.

Abstract: The iron-sulfur (Fe-S) cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into target proteins with the aid of complex machinery. In bacteria, several proteins encoded by the iscRSUA-hscBA-fdx-ORF3 cluster (isc operon) have been proposed to execute crucial tasks in the assembly of Fe-S clusters. To elucidate the in vivo function, we have undertaken a systematic mutational analysis of the genes in the Escherichia coli isc operon. In all functional tests, i.e. growth rate, nutritional requirements and activities of Fe-S enzymes, the inactivation of the iscS gene elicited the most drastic alteration. Strains with mutations in the iscU, hscB, hscA, and fdx genes also exhibited conspicuous phenotypical consequences almost identical to one another. The effect of the inactivation of iscA was small but appreciable on Fe-S enzymes. In contrast, mutants with inactivated iscR or ORF3 showed virtually no differences from wild-type cells. The requirement of iscSUA-hscBA-fdx for the assembly of Fe-S clusters was further confirmed by complementation experiments using a mutant strain in which the entire isc operon was deleted. Our findings support the conclusion that IscS, via cysteine desulfurase activity, provides the sulfur that is subsequently incorporated into Fe-S clusters by assembler machinery comprising of the iscUA-hscBA-fdx gene products. The results presented here indicate crucial roles for IscU, HscB, HscA, and Fdx as central components of the assembler machinery and also provide evidence for interactions among them.

Abstract: Iron−sulfur (Fe−S) proteins contain prosthetic groups consisting of two or more iron atoms bridged by sulfur ligands, which facilitate multiple functions, including redox activity, enzymatic function, and maintenance of structural integrity. More than 20 proteins are involved in the biosynthesis of iron−sulfur clusters in eukaryotes. Defective Fe−S cluster synthesis not only affects activities of many iron−sulfur enzymes, such as aconitase and succinate dehydrogenase, but also alters the regulation of cellular iron homeostasis, causing both mitochondrial iron overload and cytosolic iron deficiency. In this work, we review human Fe−S cluster biogenesis and human diseases that are caused by defective Fe−S cluster biogenesis. Fe−S cluster biogenesis takes place essentially in every tissue of humans, and products of human disease genes, including frataxin, GLRX5, ISCU, and ABCB7, have important roles in the process. However, the human diseases, Friedreich ataxia, glutaredoxin 5-deficient sideroblastic anemia,...