Isolation of a multiprotein complex containing cytochrome b and c1 from Neurospora crassa mitochondria by affinity chromatography on immobilized cytochrome c. Difference in the binding between ferricytochrome c and ferrocytochrome c to the multiprotein complex.
A multiprotein complex which contains in equimolar amounts two cytochromes b (Mr each about 27,000), one cytochrome c1 (Mr 31,000) and six subunits without known prosthetic groups (Mr 8000, 12,000, 14,000, 45,000, 45,000, and 50,000) has been isolated from the mitochondrial membranes of Neurospora crassa by affinity chromatography on immobilized cytochrome c. The chromatographic separation was based upon the specific binding of the complex to ferricytochrome c coupled to Sepharose and its specific release upon conversion of the coupled ferricytochrome c into ferrocytochrome c using ascorbate as a reductant. The chromatography was performed in the presence of the nonionic detergent Triton X-100 at low ionic strengths. A monodisperse preparation of the multiprotein complex was obtained which was used for binding studies with cytochrome c from Neurospora crassa, horse heart and Saccaromyces cerevisiae. At low ionic strength (20 mM Trisacetate) and slightly alkaline pH (pH 7 to 8), more than one molecule of ferricytochrome c were bound to the isolated multiprotein complex with dissociation constants below 1 x 10(-7) M. One of these bindings appeared different from the others, since its high affinity was preserved at an ionic strength at which the affinities of the other bindings decreased. Furthermore, the affinity of only this binding decreased upon reduction of cytochrome c. It is suggested that this binding is at or near the functionally active site(s) of the mulipprotein complex.
D001665:Binding Sites; D002846:Chromatography, Affinity; D003574:Cytochrome c Group; D003580:Cytochromes; D005656:Fungal Proteins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000378:metabolism; D046911:Macromolecular Substances; D008928:Mitochondria; D008970:Molecular Weight; D046912:Multiprotein Complexes; D009491:Neurospora; D009492:Neurospora crassa; D009994:Osmolar Concentration
D003574:Cytochrome c Group; D003580:Cytochromes; D005656:Fungal Proteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D007930:Leucine