Definition of the Interaction Domain and Electron Transfer Route between Cytochrome c and Cytochrome Oxidase.

The reaction between cytochrome c () and cytochrome c oxidase (CcO) was studied using horse cytochrome c derivatives labeled with ruthenium trisbipyridine at Cys 39 (Ru-39-). Flash photolysis of a 1:1 complex between Ru-39- and bovine CcO at a low ionic strength resulted in the electron transfer from photoreduced heme c to Cu with an intracomplex rate constant of = 6 × 10 s. The K13A, K72A, K86A, and K87A Ru-39- mutants had nearly the same value but bound much more weakly to bovine CcO than wild-type Ru-39-, indicating that lysines 13, 72, 86, and 87 were involved in electrostatic binding to CcO, but were not involved in the electron transfer pathway.

Chapter 28 Use of ruthenium photoreduction techniques to study electron transfer in cytochrome oxidase.

Ruthenium photoreduction methods are described to study electron transfer from cytochrome c to cytochrome c oxidase and within cytochrome oxidase. Methods are described to prepare a ruthenium cytochrome c derivative Ru-39-Cc, by labeling the single sulfhydryl group on horse K39C with (4-bromomethyl-4'methylbipyridine) (bis-bipyridine)ruthenium(II). The ruthenium complex attached to Cys-39 on the opposite side of the heme crevice does not interfere with the interaction with cytochrome oxidase.

Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase.

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.

A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase.

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e.

Single-electron photoreduction of the P(M) intermediate of cytochrome c oxidase.

The P(M)-->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 P(M) 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.

An arginine to lysine mutation in the vicinity of the heme propionates affects the redox potentials of the hemes and associated electron and proton transfer in cytochrome c oxidase.

Cytochrome c oxidase pumps protons across a membrane using energy from electron transfer and reduction of oxygen to water. It is postulated that an element of the energy transduction mechanism is the movement of protons to the vicinity of the hemes upon reduction, to favor charge neutrality. Possible sites on which protons could reside, in addition to the conserved carboxylate E286, are the propionate groups of heme a and/or heme a(3).

Direct measurement of proton release by cytochrome c oxidase in solution during the F-->O transition.

The mechanism by which electron transfer is coupled to proton pumping in cytochrome c oxidase is a major unsolved problem in molecular bioenergetics. In this work it is shown that, at least under some conditions, proton release from the enzyme occurs before proton uptake upon electron transfer to the heme/Cu active site of the enzyme. This sequence is similar to that of proton release and uptake observed for the light-activated proton pump bacteriorhodopsin.

Role of the conserved arginine pair in proton and electron transfer in cytochrome C oxidase.

A hydrogen-bonded network is observed above the hemes in all of the high-resolution crystal structures of cytochrome oxidases. It includes water and a pair of arginines, R481 and R482 (Rhodobacter sphaeroides numbering), that interact directly with heme a and the heme a(3) propionates. The hydrogen-bonded network provides potential pathways for proton release.

Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase.

The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1).

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer.

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively.