In plants, three Fe-S cluster assembly pathways exist namely SUF, ISC and CIA, involved in the maturation of iron sulfur proteins in the plastid, mitochondria and cytosol respectively. In this talk, we report on anaerobic purification of three classes of recombinant mitochondrial Fe-S cluster carrier proteins from A. thaliana, namely GRXS15, ISCA1a/2, and NFU4/5, and characterization of their Fe-S cluster content using UV-visible absorption/CD, resonance Raman, EPR, and analytical studies. GRXS15 contained trace amounts of [2Fe-2S] clusters as purified and cluster reconstitution in the presence of GSH resulted in a homogeneous [2Fe-2S] cluster-bound form. Co-expression of ISCA1a/2 resulted in samples with one [2Fe-2S] cluster per heterodimer. Unlike [2Fe-2S] cluster-bound Azotobacter vinelandii NifIscA homodimer (1), [2Fe-2S] cluster-bound ISCA1a/2 did not undergo reversible conversion to a [4Fe-4S] cluster-bound form on addition of DTT. However, cluster reconstitution using the as purified [2Fe-2S] cluster-bound ISCA1a/2 heterodimer did result in a predominantly [4Fe-4S] cluster-bound form of the ISCA1a/2 heterodimer. NFU4 and NFU5 were both purified as apo proteins, but both assembled one [4Fe-4S] cluster per homodimer after cluster reconstitution. In order to establish the role of these proteins in plant mitochondrial Fe-S cluster trafficking, cluster transfer reactions were monitored by UV-visible absorption and CD spectroscopy. The major conclusions thus far are that [2Fe-2S] cluster-bound GRXS15 is competent for assembling [4Fe-4S] clusters on the ISCA1a/2 heterodimer and [4Fe-4S]-ISCA1a/2 can transfer a [4Fe-4S] cluster to the NFU4 and NFU5 proteins. However, [2Fe-2S] cluster-bound ISCA1a/2 is not competent for the assembly of either [2Fe-2S] or [4Fe-4S] clusters on NFU4 or NFU5, and [4Fe-4S] cluster-bound NFU4 and NFU5 are not competent for the maturation of mitochondrial aconitase.
- Mapolelo, D. T., Zhang, B., Naik, S. G., Huynh, B. H., and Johnson, M. K. (2012) Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA. Biochemistry 51, 8071-8084