Department of Chemistry and Biochemistry , The Ohio State University , 100 West 18th Avenue , Columbus , Ohio 43210 , United States.
The Ohio State Biochemistry Program , The Ohio State University , 484 West 12th Avenue , Columbus , Ohio 43210 , United States.
Inorg Chem. 2019 Oct 21;58(20):13686-13695. doi: 10.1021/acs.inorgchem.9b01278. Epub 2019 Aug 22.
Iron-sulfur (Fe-S) clusters are common prosthetic groups that are found within a variety of proteins responsible for functions that include electron transfer, regulation of gene expression, and substrate binding and activation. Acquisition of a [4Fe-4S] cluster is essential for the functionality of many such roles, and dysfunctions in Fe-S cluster synthesis and trafficking often result in human disease, such as multiple mitochondrial dysfunctions syndrome. While the topic of [2Fe-2S] cluster biosynthesis and trafficking has been relatively well studied, the understanding of such processes involving [4Fe-4S] centers is less developed. Herein, we focus on the mechanism of the assembly of [4Fe-4S] clusters on two members of the aconitase family, differing also in organelle placement (mitochondrion and cytosol) and biochemical function. Two mechanistic models are evaluated by a combination of kinetic and spectroscopic models, namely, a consecutive model (I), in which two [2Fe-2S] clusters are sequentially delivered to the target, and a prereaction equilibrium model (II), in which a [4Fe-4S] cluster transiently forms on a donor protein before transfer to the target. The paper also addresses the issue of cluster nuclearity for functionally active forms of ISCU, NFU, and ISCA trafficking proteins, each of which has been postulated to exist in both [2Fe-2S] and [4Fe-4S] bound states. By the application of kinetic assays and electron paramagnetic resonance spectroscopy to examine delivery pathways from a variety of potential [2Fe-2S] donor proteins to eukaryotic forms of both aconitase and iron regulatory protein, we conclude that a consecutive model following the delivery of [2Fe-2S] clusters from NFU1 is the most likely mechanism for these target proteins.
铁硫（Fe-S）簇是常见的辅基，存在于多种负责电子传递、基因表达调控以及底物结合和激活等功能的蛋白质中。获得[4Fe-4S]簇对于许多此类功能至关重要，而 Fe-S 簇合成和运输的功能障碍通常会导致人类疾病，如多种线粒体功能障碍综合征。虽然[2Fe-2S]簇生物合成和运输的主题已经得到了相对较好的研究，但对于涉及[4Fe-4S]中心的这些过程的理解还不够发达。在此，我们重点研究了 aconitase 家族的两个成员上[4Fe-4S]簇的组装机制，这两个成员在细胞器位置（线粒体和细胞质）和生化功能上也有所不同。通过结合动力学和光谱学模型，评估了两种机制模型，即连续模型（I），其中两个[2Fe-2S]簇依次递送到靶标，以及预反应平衡模型（II），其中[4Fe-4S]簇在转移到靶标之前在供体蛋白上短暂形成。本文还解决了 ISCU、NFU 和 ISCA 转运蛋白功能活性形式的簇核性问题，这三种蛋白都被假定存在[2Fe-2S]和[4Fe-4S]结合态。通过应用动力学测定和电子顺磁共振波谱法来研究来自各种潜在[2Fe-2S]供体蛋白的递送到真核形式 aconitase 和铁调节蛋白的途径，我们得出结论，NFU1 从 NFU1 递送来[2Fe-2S]簇后，连续模型是这些靶蛋白最有可能的机制。
