Definition of cytochrome c binding domains by chemical modification: kinetics of reaction with beef mitochondrial reductase and functional organization of the respiratory chain.
An assay has been developed to study the steady-state kinetics of the reduction of cytochrome c by purified beef heart mitochondrial cytochrome c reductase (cytochrome bc(1) complex, complex III). An analogue of coenzyme Q(2) (2,3-dimethoxy-5-methyl-6-decylhydroquinone) was employed as an antimycin-sensitive reductant. The kinetics of reaction of ten different mono(4-carboxy-2,6-dinitrophenyl) derivatives of horse cytochrome c were determined. The modified proteins showed higher apparent K(m) values than the native protein and greater sensitivity to ionic strength, defining an interaction domain on cytochrome c for purified cytochrome c reductase. This interaction site is located on the front surface of the molecule (which contains the exposed heme edge) and surrounds the point at which the positive end of the dipole axis crosses the surface of the protein. The site is similar to that previously determined for mitochondrial cytochrome c oxidase and yeast cytochrome c peroxidase, suggesting that the primary interaction with redox partners is directed by the dipolar charge distribution on cytochrome c. The extensive overlapping of the interaction domains for the mitochondrial cytochrome c oxidase and reductase indicates that cytochrome c must be mobile in order to transfer electrons between them, depending on their relative positions in the membrane. Whether such mobility is necessary in intact mitochondria depends on whether the interactions with the complete membrane-bound system are the same as with the purified components.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003579:Cytochrome Reductases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D008958:Models, Molecular; D009994:Osmolar Concentration; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D003574:Cytochrome c Group; D003579:Cytochrome Reductases