[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins |
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Authors: | Tamanna Azam Jonathan Przybyla-Toscano Florence Vignols Jrmy Couturier Nicolas Rouhier Michael K Johnson |
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Institution: | 1.Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia, USA;2.Université de Lorraine, INRAE, IAM, Nancy, France;3.BPMP, Université de Montpellier, INRAE, CNRS, SupAgro, Montpellier, France |
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Abstract: | Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although 4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of 4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one 4Fe-4S]2+ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a 4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer is effective in transferring 4Fe-4S]2+ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, 4Fe-4S]2+ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective 4Fe-4S]2+ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative 4Fe-4S]2+ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client 4Fe-4S] cluster-containing proteins. |
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Keywords: | mitochondria iron-sulfur protein Arabidopsis thaliana protein-protein interaction circular dichroism Raman spectroscopy iron-sulfur cluster trafficking ISCA proteins NFU proteins |
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