Membrane crystals of ubiquinone:cytochrome c reductase from Neurospora mitochondria

Abstract

HIGH-RESOLUTION X-ray diffraction or electron microscopy studies of membrane proteins are hampered by the difficulty in obtaining crystals or periodic aggregates of the proteins. So far, only two examples of membrane proteins forming highly ordered arrays are known, the purple membrane of Halobacterium halobium¹ and a membranous preparation of cytochrome c oxidase from beef heart². Ubiquinone:cytochrome c reductase, a multiprotein enzyme, contributes about 10% of the inner mitochondrial membrane protein³. The enzyme is isolated as a dimer (molecular weight (MW) ∼550,000) and the monomeric unit comprises two b cytochromes (MW ∼30,000 each), a cytochrome c₁ (MW ∼31,000), an iron-sulphur subunit (MW ∼25,000) and six subunits without known prosthetic groups (MWs ∼50,000, 45,000, 45,000, 14,000, 12,000 and 8,000)^3,4. The enzyme catalyses the reduction of ferricytochrome c by reduced ubiquinone and conserves the energy generated by the oxidation-reduction reaction by translocating protons across the inner mitochondrial membrane⁵. Cytochrome c reductase is also of interest with respect to the genetics and biogenesis of mitochondria. The cytochrome b subunits of this enzyme belong to the small group of proteins which are translated on mitochondrial ribosomes⁶ and coded for by mitochondrial DNA⁷. We report here a method for preparing membrane crystals of ubiquinone:cytochrome c reductase isolated from Neurospora crassa.