A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc. The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1. The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.

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 k 3 = 6 × 10 4 s −1 . The K13A, K72A, K86A, and K87A Ru-39-Cc mutants had nearly the same k 3 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 k 3 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 et al. (2017) EMBO J. 36, 291−300) is not consistent with the kinetic results because the K13A mutation had no effect on k 3 . Addition of 40% ethylene glycol (as present during the crystal preparation) decreased k 3 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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