Mitochondrial Respiratory Chain Complex Ii Purified And Characterized

The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Complex II comprises of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two trans-membrane proteins (CybL and CybS), the molecular weight was 125KDa, as well as prosthetic groups required for electron transfer. Electrons are transferred from succinate to ubiquinone through the buried prosthetic groups flavin-adenine dinucleotide (FAD); the [2Fe-2S], [4Fe-4S], and [3Fe-4S] clusters; and heme b cofactors. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.A membrane protein complex, Succinate-Ubiquinone Oxidoreductase (SQR) from porcine heart has been purified by multiple frationated ammonium sulfate precipitation and Hydroxyapatite and Superdex-200 chromatogram. The purity of complex II was about 60% in the first gross purification. The following improved purification of complex II was up to 90%. It is clear to see the four subunit FAD, three iron-sulphur clusters and two small integral membrane subunits in the SDS-PAGE. In this research, SQR with inhibitors of quinone was cocrystalization.The SQR was charactered by electrophoresis and UV absorption spectroscopy. Preliminary standard reference spectra for the unliganded enzyme and for the spectral changes induced by sodium dithionite and succinate were obtained. The wavelength and intensity of the UV signal observed in agreement with results of ox-reduced state of heme b. In addition, we proved there was no other quinone binding site in both enzyme complexes by reconstruction liposome experiment. The change of SPQ fluorescence is response to internal acidification. Although it does not pump protons across the membrane as do complexes I, III, and IV, its broad species distribution reflects both its early development in the course of evolution and the selective advantage. Lastly, we examined the bisubstrates enzyme kinetic parameters and hope to study further by modification of the SQR.