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

Abstract

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.

New insights into the proton pumping mechanism of ba3 cytochrome c oxidase: the functions of key residues and water

“…Analyzing the conformation changes in MD simulation trajectories of His376Phe and His376Tyr, it was discovered that there is consistently a hydrogen-bonded water molecule between PRAa 3 and PRDa 3 . 71 This leads to two distinct possible paths for the proton transfer, one of which involves the stable water molecule between PRAa 3 and PRDa 3 (Fig. 6(D) and (F)), while the other does not (Fig.…”

Conformational and mechanical stability of the isolated large subunit of membrane-bound [NiFe]-hydrogenase from Cupriavidus necator

“…The initial protonation pattern ( Supplementary Table S1 ; SI) was determined based on the initial model [HoxG from the crystal structure 3RGW ( Fritsch et al, 2011 ) with modelled N-terminal Strep-tag II as a random coil, Figure 1 ], by computing pK A values with Karlsberg2 + (KB2 + ; Rabenstein and Knapp, 2001 ; Kieseritzky and Knapp, 2008 ; Meyer and Knapp, 2015 ), as in previous applications ( Wolf et al, 2020 ; Dragelj et al, 2021a , b ; Batebi et al, 2018 ). The pK A values have been computed in the range from −10.00 to 20.00, not only for the starting model but also for the structures from GaMD trajectories obtained every 2 ns.…”

The Catalytic Reaction of Cytochrome c Oxidase Probed by In Situ Gas Titrations and FTIR Difference Spectroscopy

“…Following this interpretation, the water cavity above the heme cofactors [46,57] would possibly connect their propionic acid moieties through a hydrogen-bonding network. It has been suggested that the propionic acids of heme a3 "store" a proton [101], making the whole hydrogen-bonding network a candidate for the PLS. A similar concept has been discussed for the transmembrane proton pump bacteriorhodopsin, where two glutamic acid residues share a proton to form a "intermolecular proton bond" acting as the proton release group [102].…”