Characterizing the proton loading site in cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, Cu(B), Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.

Keywords: MCCE; bioenergetics; pKa; proton transfer.

Fig. 1.

Key groups in CcO. The redox and protonation states of heme a, a₃, Cu_B, Y288, and E286 are defined to change through the reaction cycle (Fig. 2 and SI Appendix, Fig. S2 and Table S1). Cu_A is always oxidized here. PRA and PRD, which are the propionic acids of heme a and a₃, are allowed to come to equilibrium with the imposed charges in each substate.

Fig. 2.

(Upper) The four redox states of the BNC. In R state O₂ binds the reduced BNC. Each state is separated by the addition of one electron from heme a and one proton from E286 to the BNC. Red, reduced; green, more oxidized; blue, most oxidized. (Lower) The substates for the F state. The sequence shown starts at the end of the R state (R6) with heme a₃ and Cu_B reduced, O₂ bound in the BNC, and E286 having released the proton to load the PLS. Electron transfer from heme a to the BNC triggers reduction of O₂ to generate the P_R state (F1 and F2 here). E286 is then protonated via the D-channel. The proton is transferred from E286 to the BNC. In the F state the proton acceptor is the hydroxyl ligand to Cu_B. E286 is then reprotonated and heme a is reduced by cytochrome c via Cu_A. Then E286 loses a proton, which should be loaded into the PLS for pumping. This substate (F6) is now prepared for the next electron transfer to form the O1 substate as shown. The transitions i, ii, iv, and v are described in the text (5). Transition iii is proton release from the PLS. In the calculations, proton release is not fixed in any step; rather, it results from the change in the electrostatic environment, so this step is not explicitly included in the reaction cycle. Rather, the number of protons bound to the PLS is monitored in all substates to identify when protons are released. In calculations of transition v, a proton is removed from E286 without designating the proton acceptor. See SI Appendix, Fig. S2 and Table S1 for a description of the entire reaction cycle.

Fig. 3.

(A) The charge change in the CcO 1M56 structure as it goes through the defined substates reaction cycle (Fig. 2). Each summed charge is provided relative to the charge in the R4 substate where the PLS has the fewest protons bound. R4 is formed by reprotonation of E286 with heme a oxidized. Red line, charge imposed on BNC (the summed charge of Y288, heme a₃, Cu_B, and their ligands), E286, and heme a. Green line, net charge of the PLS. Purple line, charge of the non-PLS. Black line, total charge of the protein. (B) Substate cycle without fixing E286 in 1M56a. Now only substates X2, X4, and X5 are imposed for each of the four redox states (brown circles). E286 remains neutral in all substates. The imposed charge, which now includes heme a, a₃, Cu_B, and Y288 only (red line), varies from 0 to 1. The sum charge on the PLS with (green line)/without (brown line) E286 fixed is shown.

Fig. 4.

Dependence of the PLS proton uptake on the proton affinity of heme a₃ acids (${\text{pK}\prime }_7$)_. The change in PLS protonation in the R1 (●) and E1 (○) substates relative to that found in the R4 substate, which has the fewest protons bound. The higher ${\text{pK}\prime }_7$ of heme a₃ acids is used for each CcO structure in R1 or E1 substate. The linear regression line has an R² of 0.729.