Definition of the Interaction Domain for Cytochromecon the Cytochromebc1Complex

Hua Tian, Robert Sadoski, Li Zhang, Chang-An Yu, Linda Yu, Bill Durham, Francis Millett
2000 Journal of Biological Chemistry  
The interaction domain for cytochrome c on the cytochrome bc 1 complex was studied using a series of Rhodobacter sphaeroides cytochrome bc 1 mutants in which acidic residues on the surface of cytochrome c 1 were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc 1 at low ionic strength resulted in
more » ... lectron transfer from photoreduced heme c to cytochrome c 1 with a rate constant of k et ‫؍‬ 6 ؋ 10 4 s ؊1 . Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k et values as well as significantly higher equilibrium dissociation constants and steadystate K m values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c 1 are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c 1 direct the diffusion and binding of cytochrome c from the intramembrane space.
doi:10.1074/jbc.275.13.9587 pmid:10734109 fatcat:gkpj454td5cuxfjuzwsmumi72e