Design of a Ruthenium-Cytochrome c Derivative to Measure Electron Transfer to the Initial Acceptor in Cytochrome c Oxidase

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c

Accelerated Evolution of Cytochrome c in Higher Primates, and Regulation of the Reaction between Cytochrome c and Cytochrome Oxidase by Phosphorylation

Cytochrome c (Cc) underwent accelerated evolution from the stem of the anthropoid primates to humans. Of the 11 amino acid changes that occurred from horse Cc to human Cc, five were at Cc residues near the binding site of the Cc:CcO complex. Single-point mutants of horse and human Cc were made at each of these positions. The Cc:CcO dissociation constant KD of the horse mutants decreased in the order: T89E > native horse Cc > V11I Cc > Q12M > D50A > A83V > native human. The largest effect was observed for the mutants at residue 50, where the horse Cc D50A mutant decreased KD from 28.4 to 11.8 μM, and the human Cc A50D increased KD from 4.7 to 15.7 μM. To investigate the role of Cc phosphorylation in regulating the reaction with CcO, phosphomimetic human Cc mutants were prepared. The Cc T28E, S47E, and Y48E mutants increased the dissociation rate constant kd, decreased the formation rate constant kf, and increased the equilibrium dissociation constant KD of the Cc:CcO complex. These studies indicate that phosphorylation of these residues plays an important role in regulating mitochondrial electron transport and membrane potential ΔΨ

Single-electron photoreduction of the PM intermediate of cytochrome c oxidase

AbstractThe PM→F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The PM state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F→O transition, but is significantly slower than formation of the F state from the PR intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70–90 μs). The ∼0.3 ms PM→F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the PM→F transition is generated much later, with a time constant of 1.3 ms. In addition, the PM→F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms