The reaction between cytochrome c (Cc) and cytochrome c oxidase (CcO) was studied using horse cytochrome c derivatives labeled with ruthenium trisbipyridine at Cys 39 (Ru-39-Cc). Flash photolysis of a 1:1 complex between Ru-39-Cc and bovine CcO at a low ionic strength resulted in the electron transfer from photoreduced heme c to Cu_A with an intracomplex rate constant of k₃ = 6 × 10⁴ s^-1. The K13A, K72A, K86A, and K87A Ru-39-Cc mutants had nearly the same k₃ value but bound much more weakly to bovine CcO than wild-type Ru-39-Cc, indicating that lysines 13, 72, 86, and 87 were involved in electrostatic binding to CcO, but were not involved in the electron transfer pathway. The Rhodobacter sphaeroides (Rs) W143F mutant (bovine W104) caused a 450-fold decrease in k₃ but did not affect the binding strength with CcO or the redox potential of Cu_A. These results are consistent with a computational model for Cc-CcO (Roberts and Pique ( 1999 ) J. Biol. Chem. 274 , 38051 - 38060 ) with the following electron transfer pathway: heme c → CcO-W104 → CcO-M207 → Cu_A. A crystal structure for the Cc-CcO complex with the proposed electron transfer pathway heme c → Cc-C14 → Cc-K13 → CcO-Y105 → CcO-M207 → Cu_A ( S. Shimada ( 2017 ) EMBO J. 36 , 291 - 300 ) is not consistent with the kinetic results because the K13A mutation had no effect on k₃. Addition of 40% ethylene glycol (as present during the crystal preparation) decreased k₃ significantly, indicating that it affected the conformation of the complex. This may explain the discrepancy between the current results and the crystallographic structure.

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