期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 21, 期 23, 页码 -出版社
MDPI
DOI: 10.3390/ijms21239237
关键词
mitochondria; iron-sulfur protein; Arabidopsis thaliana; protein-protein interaction; circular dichroism; Raman spectroscopy; iron-sulfur cluster trafficking; glutaredoxin; ISCA proteins
资金
- National Institutes of Health [R37GM62524]
- Agence Nationale de la Recherche as part of the Investissements d'Avenir program [ANR-11-LABX-0002-01, ANR-15-IDEX-04-LUE]
- [ANR-2013-BSV6-0002-01]
Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S](2+) cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S](2+) cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S](2+) cluster assembly on mitochondrial ferredoxin and [4Fe-4S](2+) cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S](2+) clusters, via two-electron reductive coupling of two [2Fe-2S](2+) clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S](2+) to [4Fe-4S](2+) cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.
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