Cytochrome c oxidase

Approximately up to ten intermediates of the catalytic cycle have been identified. The intermediates have distinct spectroscopical properties and defined with letter abbreviations. Each letter abbreviation reflects only the state of the binuclear site and do not specify the redox states of heme a and CuA.

Catalytic cycle of cytochrome c oxidase

Catalytic cycle of cytochrome c oxidase

  • Fully oxidized O-state. The enzyme after purification is in the fully-oxidized "as prepared" state, where all redox centers of the protein are oxidized and the enzyme cannot react with dioxygen.

  • One-electron reduced E-state. In the E state an electron is shared between heme a3 and CuB in the binuclear center. Due to extremely slow proton uptake to the binuclear center for charge compensation formation of the E state takes seconds. Since the complete cycle of CcO occurs in a few milliseconds, it is rather unlikely that the E state is a natural state during catalytic turnover of the oxidase.

  • Reduced R-state. The R state is the state which is capable of dioxygen binding and can have from 2 (mixed-valence state) to 4 (fully-reduced state) electrons in the redox centers.

  • Ferrous-oxy A intermediate. The reduced binuclear site is rapidly react with dioxygen producing the so-called compound A. At room temperature compound A is formed with a time constant of 8 μs at 1 mM of oxygen.

  • Peroxy intermediate, PM. The PM intermediate is formed when the reation starts from the two-electron reduced state of the enzyme. PM is stable and the reaction stops here unless an additional electron enters the enzyme. This compound was named "peroxy" because it was thought that the oxygen-oxygen bond was still intact, and heme a3 had a peroxy structure: Fe-O-O. In reality the O-O bond is already broken in PM and heme a3 is in the oxo-ferryl state. Cleavage of the O-O bond requires simultaneous transfer of four electrons to the molecule of dioxygen. Three of these electrons are donated from the metals of the binuclear center: one from CuB and two from the heme a3 iron. The fourth electron is donated from one of the amino-acid residues in the proximity of the catalytic site. Presumably by the cross-linked tyrosine-histidine dimer. In addition to electron transfer, O-O bond cleavage requires delivery of a proton, which is most likely also borrowed from the cross-linked dimer, producing a neutral tyrosine radical.

  • Peroxy intermediate, PR. When the oxygen reacts with the fully-reduced oxidase, compound A relaxes in about 30-40 μs into another unstable peroxy intermediate (PR). Similar to PM the O-O bond is also broken in PR. The fourth required electron is donated not from tyrosine but from heme a. However the required proton is still picked up from the cross-linked tyrosine with formation of the deprotonated tyrosinate.

  • Ferryl-oxo intermediate F. From the structural point of view one of the differences between F and PR is an extra proton in the catalytic center in the former intermediate. This proton is taken up from the N-side of the membrane via the D-pathway. The final destination of the proton during the P->F transition is uncertain but two possible candidates can be proposed: Tyr-O-, or the hydroxyl group on CuB. Formation of F is also coupled with electron equilibration between CuA and heme a.

  • Fully-Oxidized High-Energy state, OH. The OH state appears from F with a time constant of 1-2 ms and requires delivery of both an electron and a proton to the catalytic site. While the electron migrates from the CuA/heme a couple, the proton is taken up via the D-pathway similar to formation of F. The fully-oxidized OH state is referred to as a "high-energy" state implying that the energy released in the redox reactions of the oxidative part of the catalytic cycle is conserved in this intermediate, and will be used during the next transitions of the cycle for proton pumping. The OH state is not stable and relaxes into the low energy O state (incapable of pumping protons upon reduction) possibly by a protonation of a hydroxyl to water, or tyrosinate to tyrosine.

  • One-electron reduced EH. The EH state is formed by a single-electron reduction of OH. As a result of the reaction the electron resides on CuB. In contrast to E, the EH state is capable to proton pumping upon reduction.

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