Beating Heart of Cytochrome c Oxidase: The Shared Proton of Heme a3 Propionates

Cytochrome c oxidase (CcO) pumps protons from the N-side to the P-side and consumes electrons from the P-side of the mitochondrial membrane driven by energy gained from reduction of dioxygen to water. ATP synthesis uses the resulting proton gradient and electrostatic potential difference. Since the distance a proton travels through CcO is too large for a one-step transfer process, proton-loading sites (PLS) that can carry protons transiently are necessary. One specific pump-active PLS couples to the redox reaction, thus energizing the proton to move across the membrane against electric potential and proton gradient. The PLS should also prevent proton backflow. Therefore, the propionates of the two redox-active hemes in CcO were suggested as PLS candidates although, according to CcO crystal structures, none of the four propionates can be protonated on account of strong H-bonds. Here, we show that modeling the local structure around heme a(3) propionates enhances significantly their capability of carrying a proton jointly. This was not possible for the propionates of heme a. The modeled structures are stable in molecular dynamics simulations (MDS) and are energetically similar to the crystal structure. Precise electrostatic energy computations of MDS data are used to estimate the pK(A) values of all titratable residues in CcO. For the modeled structures, the heme a(3) propionates have pK(A) values high enough to host a proton transiently but not too high to fix the proton permanently. The change in pK(A) throughout the redox reaction is sufficient to push the proton to the P-side of the membrane and to provide the protons with the necessary amount of energy for ATP synthesis.

Automated summary

Cytochrome c oxidase pumps protons and consumes electrons on the mitochondrial membrane to create energy for ATP synthesis. Proton-loading sites are crucial for transient proton transport and prevention of backflow. Modeling heme a(3) propionates enhances their capability to carry protons and potentially contributes to the energy transfer mechanism for ATP synthesis.