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

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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.