Poster: Enzymology

Abs # 236: A HEAT-repeats containing protein, IaiH, stabilizes the iron-sulfur cluster bound to the cyanobacterial IscA homologue, IscA2

Presenter:	Morimoto, Kozo , kozo@protein.osaka-u.ac.jp
Authors	Morimoto, Kozo  (A)   Shusei, Sato  (B)   Satoshi, Tabata  (B)   Masato, Nakai  (A)
Affiliations:	(A): Institute for Protein Research, Osaka University (B): Kazusa DNA Research Institute, Japan

IscA homologues are involved in iron-sulfur cluster biosynthesis. In the non-nitrogen-fixing cyanobacterium, Synechocystis PCC 6803, there are two IscA homologues, SLR1417 and SLR1565 (designated IscA1 and IscA2), of which only IscA2 exists as a protein complex with the HEAT-repeat-containing protein, SLR1098 (IaiH). We observed that the absorption spectrum of recombinant IscA2/IaiH complex resembled that of IscA2 alone, although was sharper. In the presence of dithiothreitol, the [2Fe-2S] cluster of IscA2 alone, but not of the IscA2/IaiH complex, became reductively labile on the addition of sodium dithionite. This implies that the IscA2 moiety of the [2Fe-2S] cluster is stabilized by the presence of IaiH. The [2Fe-2S] cluster of the IscA2/IaiH complex was destabilized by sodium dithionite in the absence of dithiothreitol, suggesting that the in vivo stability of the iron-sulfur cluster in the IscA2/IaiH complex is influenced by the redox state of cellular thiols. When any one of three conserved cysteine residues in IscA2, potential ligands for the [2Fe-2S] cluster, was substituted with serine, the level of assembled [2Fe-2S] cluster and protein complex was significantly reduced in E. coli cells. The cysteine mutated IscA2/IaiH complexes that were present all contained a [2Fe-2S]-like cluster suggesting that the assembly of a stable iron-sulfur cluster bound to IscA2 is required for efficient and stable complex formation. Truncated IaiH proteins were analyzed using the yeast two-hybrid assay to identify the essential domain of IaiH that interacts physically with IscA2. At least 2 of the 5 N-terminal HEAT repeats of IaiH were found to be required for interaction with IscA2.