Zafreen Lala, Walker-Kopp Nancy, Huang Li-Shar, Berry Edward
Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
Arch Biochem Biophys. 2016 Aug 15;604:47-56. doi: 10.1016/j.abb.2016.06.006. Epub 2016 Jun 11.
Mitochondrial Complex II (Succinate: ubiquinone oxidoreductase) has a covalently bound FAD cofactor in its largest subunit (SDHA), which accepts electrons from oxidation of succinate during catalysis. The mechanism of flavin attachment, and factors involved, have not been fully elucidated. The recent report of an assembly factor SDH5 (SDHAF2, SDHE) required for flavinylation (Hao et al., 2009 Science 325, 1139-1142) raises the prospect of achieving flavinylation in a completely defined system, which would facilitate elucidation of the precise role played by SDH5 and other factors. At this time that goal has not been achieved, and the actual function of SDH5 is still unknown. We have developed a procedure for in-vitro flavinylation of recombinant human apo-SDHA, immobilized on Ni-IMAC resin by a His tag, in a chemically defined medium. In this system flavinylation has a pH optimum of 6.5 and is completely dependent on added SDH5. The results suggest that FAD interacts noncovalently with SDHA in the absence of SDH5. This system will be useful in understanding the process of flavinylation of SDHA and the role of SDH5 in this process.
线粒体复合物II（琥珀酸：泛醌氧化还原酶）在其最大亚基（SDHA）中具有共价结合的FAD辅因子，在催化过程中该辅因子接受来自琥珀酸氧化的电子。黄素附着的机制以及相关因素尚未完全阐明。最近有报道称黄素化需要组装因子SDH5（SDHAF2，SDHE）（Hao等人，《科学》，2009年，第325卷，第1139 - 1142页），这为在完全确定的系统中实现黄素化带来了希望，这将有助于阐明SDH5和其他因素所起的精确作用。目前这一目标尚未实现，SDH5的实际功能仍然未知。我们开发了一种程序，用于在化学定义的培养基中，对通过His标签固定在镍离子亲和层析树脂上的重组人脱辅基SDHA进行体外黄素化。在这个系统中，黄素化的最适pH为6.5，并且完全依赖于添加的SDH5。结果表明，在没有SDH5的情况下，FAD与SDHA非共价相互作用。该系统将有助于理解SDHA的黄素化过程以及SDH5在此过程中的作用。
