Arabidopsis thaliana Nfu2 Accommodates [2Fe-2S] or [4Fe-4S] Clusters and Is Competent for in Vitro Maturation of Chloroplast [2Fe-2S] and [4Fe-4S] Cluster-Containing Proteins

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Gao, Huanyao | Subramanian, Sowmya | Couturier, Jérémy | Naik, Sunil G. | Kim, Sung-Kun | Leustek, Thomas | Knaff, David B. | Wu, Hui-Chen | Vignols, Florence | Huynh, Boi Hanh | Rouhier, Nicolas | Johnson, Michael K.

Edité par CCSD ; American Chemical Society -

Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with the deletion of the gene encoding a chloroplast-specific Nfii-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV visible absorption and circular dichroism and resonance Raman and Mossbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S](2) and [4Fe-4S](2+) clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S]2+ cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4Sr](+2) cluster donor for adenosine 5'-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfia but not with Mil and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.

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