Studies on the structure and function relationship offumarate redudase from adut Ascais suum

在真核生物中，复合体II定位于线粒体膜内，一般由4个亚基组成(两个亲水性亚基Fp和Lp，以及两个疏水性膜锚定亚基CybL和CybS)。该酶催化琥珀酸氧化为富马酸，同时在有氧呼吸过程中将泛醌还原为泛喹酚。另一方面，来自猪蛔虫成虫的化合物II，生活在厌氧的小肠中，催化反相反应，富马酸还原为琥珀酸，同时氧化对苯二酚为对苯二酚。在本研究中，对化合物II进行了纯化、结晶并测定了其三维结构。在八乙二醇单十二烷基醚和十二烷基麦芽糖苷存在下，以PEG3350为沉淀剂，用透析法得到了适合于X-射线结构分析的晶体。X射线衍射数据是在BL44XU(Spring-8，日本)光束线上以2.8A的分辨率收集的。用猪配合物II的精化坐标用分子置换方法进行了结构计算，精化后的最终结构的R因子为25.0%(R-Free=28.8%)。猪链霉菌成体复合体II的结构与大肠杆菌、猪和禽类等数据的结构基本相同，特别是位于罗丹酚和富马酸结合位点之间的辅因子(一个FAD和三个铁硫簇)被许多物种的复合体IIS中保守的氨基酸残基包围。此外，与FP亚基结合的富马酸面向FAD异四氧嘧啶环并呈扭曲构象。Ip、CybL和CybS亚基形成的柔酚结合部位也主要由保守的氨基酸残基构成。基于猪链霉菌线粒体复合体II的三维结构，可以从结构上深入了解苯二酚氧化成苯二酚的机理。

In eukaryotes, complex II is localized in the inner mitochondrial membrane, and is generally composed of 4 subunits (two hydrophilic subunits, Fp and lp, and two hydrophobic membrane anchoring subunits, CybL and CybS). The enzyme catalyzes the oxidation of succinate to fumarate in conjunction with the reduction of ubiquinone to ubiquinol during aerobic respiration. On the other hand, Complex II from adult Ascaris suum, living in anaerobic small intestine, catalyzes the reverse reaction, the reduction of fumarate to succinate in conjunction with the oxidation of rhodoquinol to rhodoquinone. In this study, Complex II was purified, crystallized and its three-dimensional structure was determined. Crystals suitable for X-ray structure analysis were obtained by the dialysis method using PEG3350 as a precipitant in the presence of octaethyleneglycol monododecyl ether and dodecy maltoside. X-ray diffraction data were collected to a resolution of 2.8 A at beamline BL44XU (Spring-8, Japan). The structure was solved by the molecular replacement method using the refined coordinates of the porcine Complex II. After refinement, the final structure gave R-factor of 25.0%(R-free=28.8%). The structure of the adult A. suum Complex II is essentially identical to those determined to data such as from Escherichia coli, porcine and avian etc. Especially, cofactors (one FAD and three iron-sulfur clusters) located between rhodoquinol and fumarate binding sites are surrounded by amino acid residues well conserved in Complex Iis from many species. Further, fumarate bound to the Fp subunit faces to the FAD isoalloxazine ring and takes twisted conformation. The rhodoquinone binding site formed by the Ip, CybL and CybS subunits is also mainly constructed by well conserved amino acid residues. Based on the three-dimensional structure of Complex II from A. suum mitochondria, structural insight into the mechanism of the oxidation of rhodoquinol to rhodoquinone could be obtained.