Definition of the Interaction Domain for Cytochrome c on the Cytochrome bc sub(1) Complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc sub(1) surface mutants

The interaction domain for cytochrome c on the cytochrome bc sub(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc sub(1) mutants in which acidic residues on the surface of cytochrome c sub(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 sub(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c sub(1) with a rate constant of k sub(et) = 6 x 10 super(4) s super(-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 sub(et) values as well as significantly higher equilibrium dissociation constants and steady-state K sub(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 sub(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c sub(1) direct the diffusion and binding of cytochrome c from the intramembrane space.