Dos Santos Patricia C
Wake Forest University, Winston-Salem, NC, United States.
Methods Enzymol. 2017;595:185-212. doi: 10.1016/bs.mie.2017.07.009. Epub 2017 Aug 18.
Complexes of iron and sulfur (Fe-S clusters) are widely distributed in nature and participate in essential biochemical reactions. The biological formation of Fe-S clusters involves dedicated pathways responsible for the mobilization of sulfur, the assembly of Fe-S clusters, and the transfer of these clusters to target proteins. Genomic analysis of Bacillus subtilis and other Gram-positive bacteria indicated the presence of only one Fe-S cluster biosynthesis pathway, which is distinct in number of components and organization from previously studied systems. B. subtilis has been used as a model system for the characterization of cysteine desulfurases responsible for sulfur mobilization reactions in the biogenesis of Fe-S clusters and other sulfur-containing cofactors. Cysteine desulfurases catalyze the cleavage of the C-S bond from the amino acid cysteine and subsequent transfer of sulfur to acceptor molecules. These reactions can be monitored by the rate of alanine formation, the first product in the reaction, and sulfide formation, a byproduct of reactions performed under reducing conditions. The assembly of Fe-S clusters on protein scaffolds and the transfer of these clusters to target acceptors are determined through a combination of spectroscopic methods probing the rate of cluster assembly and transfer. This chapter provides a description of reactions promoting the assembly of Fe-S clusters in bacteria as well as methods used to study functions of each biosynthetic component and identify mechanistic differences employed by these enzymes across different pathways.
铁硫簇（Fe-S簇）复合物在自然界中广泛分布，并参与重要的生化反应。Fe-S簇的生物合成涉及专门的途径，负责硫的动员、Fe-S簇的组装以及将这些簇转移到目标蛋白质上。枯草芽孢杆菌和其他革兰氏阳性细菌的基因组分析表明，仅存在一条Fe-S簇生物合成途径，其在组成成分数量和组织方式上与先前研究的系统不同。枯草芽孢杆菌已被用作模型系统，用于表征负责Fe-S簇生物合成及其他含硫辅因子中硫动员反应的半胱氨酸脱硫酶。半胱氨酸脱硫酶催化从氨基酸半胱氨酸上裂解C-S键，并随后将硫转移到受体分子上。这些反应可以通过反应的第一个产物丙氨酸的生成速率以及在还原条件下进行反应的副产物硫化物的生成速率来监测。通过探测簇组装和转移速率的光谱方法相结合，来确定Fe-S簇在蛋白质支架上的组装以及这些簇向目标受体的转移。本章描述了促进细菌中Fe-S簇组装的反应，以及用于研究每个生物合成成分功能并识别这些酶在不同途径中所采用机制差异的方法。
