TitleArabidopsis 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.
Publication TypeJournal Article
Year of Publication2013
AuthorsGao, H, Subramanian, S, Couturier, J, Naik, SG, Kim, S-K, Leustek, T, Knaff, DB, Wu, H-C, Vignols, F, Huynh, BHanh, Rouhier, N, Johnson, MK
JournalBiochemistry
Volume52
Issue38
Pagination6633-45
Date Published2013 Sep 24
ISSN1520-4995
KeywordsArabidopsis Proteins, Chloroplasts, Endonucleases, Glutaredoxins, Iron-Sulfur Proteins, Oxidoreductases Acting on Sulfur Group Donors, Spectrophotometry, Ultraviolet, Spectrum Analysis, Raman
Abstract

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 Nfu-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 Mössbauer 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-4S](2+) cluster donor for adenosine 5'-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 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.

DOI10.1021/bi4007622
Alternate JournalBiochemistry
PubMed ID24032747
PubMed Central IDPMC3819817
Grant ListGM47295 / GM / NIGMS NIH HHS / United States
GM62524 / GM / NIGMS NIH HHS / United States
R01 GM062524 / GM / NIGMS NIH HHS / United States
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