Cytochrome c oxidase
The chemical reaction of oxygen reduction to water, which triggers proton translocation across the membrane, occurs at the binuclear center in the middle of the protein and requires both delivery of substrates (i.e. electrons, protons, and oxygen) and release of products (water). All these aforementioned reactants, which are necessary for the functioning of the oxidase, are transported to the catalytic site through specialized pathways.
The reduction of dioxygen to water requires four electrons. These electrons are donated one by one from water-soluble cytochrome c, which serves as one-electron mediator between the cytochrome bc1-complex and CcO. As soon as cytochrome c binds to the oxidase, the electron rapidly, with time constant of about 10-15 μs is transferred to the primary electron acceptor - the bimetallic copper center, CuA.
From CuA the electron in faster than 10 μs equilibrates with heme a. The centre-to-centre distance between CuA and the iron atom of heme a is 19.5 Å and this is only 2.6 Å closer than the distance between CuA and the iron atom of heme a3 (22.1 Å). However despite such similarity in distances, the preference in electron transfer from CuA is completely biased towards heme a, because the midpoint redox potential of heme a3 without protonation is far too low to permit electron transfer; thus the rate of heme a3 reduction is limited by slow proton uptake for charge compensation.
Heme a serves as a donor of electrons to the heme a3-CuB center. The minimal edge-to-edge distance between the hemes is about 4.7 Å. Rate-limiting arrival of a proton to the binuclear center increases midpoint potential of binuclear center and insures electron equilibration between the hemes on nanosecond timescale.
Since the redox centers of the oxidase are buried within the protein they have no direct contact with the aqueous phase. Since the unstructured protein medium itself cannot facilitate efficient proton delivery towards the binuclear center the oxidase has specialized proton-conductive structures. It is proposed that these structures are based on chains of hydrogen bonds between hydrogen-bonding protein side groups (polar and/or protonatable) and water molecules. The proton transfer inside the protein occurs by a Grotthuss mechanism.
At least two proton-conductive channels have been identified in CcO. Both channels are situated in subunit I and lead from the N-side of the membrane towards the catalytic center of the oxidase.
One is the K-pathway, named after the highly conserved lysine 354 (P. denitrificans numbering), which is situated approximately halfway through the channel. This pathway starts with either SerI291 or GluII78 and continues through conserved residues LysI354, ThrI351, towards the hydroxyethyl farnesyl side chain of heme a3 and the cross-linked tyrosine-histidine dimer at the catalytic center.
The other channel, named D after the highly conserved AspI124 at the surface of the enzyme on the N-side. AspI124 together with ThrI203 and AsnI199 form a mouth that leads via a group of polar residues and crystallographically identified bound water molecules to GluI278, which is an important switch residue for proton pumping.
The D-channel is involved in the uptake of all four "pumped" protons and two "chemical" protons used in the oxidative part of the catalytic cycle, while the K-channel is responsible for the uptake of another two "chemical" protons during the reductive part of the cycle. In addition, LysI354 may also be involved in the oxidative part of the cycle providing charge compensation upon electron transfer from heme a to the binuclear site and formation of the P intermediate.
As a small, uncharged molecule, dioxygen can easily permeate membranes and on a first sight should be able to reach the catalytic site of the oxidase even without any specific route through loosely packed regions using conformational fluctuations of the protein. However, the rate of such uncontrolled diffusion is slow, and most likely insufficient to maintain normal catalytic activity of the oxidase. Indeed from one (in P. denitrificans) to three (in bovine) highly hydrophobic passages from the middle of the membrane bilayer, where oxygen is concentrated, towards the active site were revealed. Interestingly, even a single amino-acid mutation can dramatically influence the binding of oxygen, causing partial or even complete inhibition of the catalysis.
© Ilya Belevich & Michael Verkhovsky