Electric fields control water-gated proton transfer in cytochrome c oxidase

Abstract

Aerobic life is powered by membrane-bound enzymes that catalyze the transfer of electrons to oxygen and protons across a biological membrane. Cytochrome c oxidase (CcO) functions as a terminal electron acceptor in mitochondrial and bacterial respiratory chains, driving cellular respiration and transducing the free energy from O₂ reduction into proton pumping. Here we show that CcO creates orientated electric fields around a nonpolar cavity next to the active site, establishing a molecular switch that directs the protons along distinct pathways. By combining large-scale quantum chemical density functional theory (DFT) calculations with hybrid quantum mechanics/molecular mechanics (QM/MM) simulations and atomistic molecular dynamics (MD) explorations, we find that reduction of the electron donor, heme a, leads to dissociation of an arginine (Arg438)-heme a₃ D-propionate ion-pair. This ion-pair dissociation creates a strong electric field of up to 1 V Å^-1 along a water-mediated proton array leading to a transient proton loading site (PLS) near the active site. Protonation of the PLS triggers the reduction of the active site, which in turn aligns the electric field vectors along a second, "chemical," proton pathway. We find a linear energy relationship of the proton transfer barrier with the electric field strength that explains the effectivity of the gating process. Our mechanism shows distinct similarities to principles also found in other energy-converting enzymes, suggesting that orientated electric fields generally control enzyme catalysis.

Keywords: PCET; QM/MM; bioenergetics; heme-copper oxidases; molecular simulations.

Fig. 1.

Structure and function of CcO. (A) CcO from Bos taurus (PDB ID: 1V54) in its membrane environment (water and ions are omitted for visual clarity). CcO catalyzes the reduction of molecular O₂ to water with four sequential electron transfer steps from cytochrome c that couple to the transfer of four protons across the membrane. Protons enter the active site through the D- and K-channels. (B) Catalytic cycle of CcO: pumped protons are represented in black, chemical protons from D-channel in blue, and chemical protons from K-channel in red. (C) The CcO nonpolar cavity around the active site is formed by heme a, involved in electron transfer, and the BNC comprising heme a₃ and Cu_B, where the O₂ reduction takes place. This model shows the proton transfer pathways of a pumped proton (from Glu242 to D-propionate, in red) and a chemical proton (from Glu242 to the BNC, in blue).

Fig. 2.

Energetics and dynamics of the proton transfer along the pumping pathway. (A) Dynamics of the Arg438/heme a₃ D-propionate ion-pair in the P_M state with heme a oxidized (red) and heme a reduced (blue). The ion-pair conformation is represented by the reaction coordinate R, defined as the difference of distances of Arg438 to heme a₃ D-propionate (r₁) and Arg438 to heme a D-propionate (r₂). Reduction of heme a induces opening of the ion-pair. (B) (Left) Energetics of the proton transfer reaction from Glu242 to D-propionate (pumping pathway) in the P_M state with heme a oxidized/ion-pair closed (red) and heme a reduced/ion-pair open (blue). (Right) Structure of the protonated water intermediates along the proton wire, connecting Glu242 with the D-propionate. (C) Electric field vectors along the pumping pathway. Overall location (Left) and the resulting electric field difference vector [ΔE_f = E_f(a^red/R438 open) − E_f(a^ox/R438 closed)] points in the direction of D-propionate (Right). See SI Appendix, Fig. S4 for detailed representations of individual electric field vectors. (D) Electric field strength along the vector connecting the water oxygen atoms along the proton transfer pathway. (E) The proton transfer barrier versus electric field at position of the protonated water cluster, calculated along the ion-pair dissociation scan, showing linear dependence between the barrier and field (see also SI Appendix, Fig. S3).

Fig. 3.

Energetics of the proton transfer reaction along the chemical pathway. (A) Energy profiles for the chemical proton transfer process with oxidized (BNC^ox/P_M) and reduced (BNC^red/P_R) BNC and a protonated PLSH. (Right) Structure of the protonated water wire connecting Glu242 to Cu_B at the transition state. (B) Electric field vectors along the chemical proton transfer pathway. (Left) Overall location. (Right) Resulting electric field difference vector [ΔE_f = E_f(BNC^red) − E_f(BNC^ox)] is pointing in the direction of the BNC. See SI Appendix, Fig. S5 for detailed representations of individual electric field vectors. (C) Electric field strengths along the vector connecting the water oxygen atoms along the chemical proton pathway.

Fig. 4.

Proposed gating principles in CcO. Reduced cofactors are colored in gray, and protonated PLSH is represented by a red ellipse. (A) Electron transfer (eT) from cytochrome c to heme a creates an electric field (E_f) in the direction of heme a that causes dissociation of Arg438 from heme a₃. (B) The Arg438 flip leaves the negative D-propionate exposed that creates an electric field along the Glu242→D-propionate direction that induces the transfer of a proton toward the PLS. (C) The PLSH state increases the electron affinity of the BNC. Electron transfer from heme a to the BNC creates an electric field toward Cu_B facilitating the proton transfer from Glu242 (with prior Glu242 reprotonation from the D-channel). (D) Heme a oxidation causes Arg438 to close back toward the PLSH that pushes the stored proton to the P-side bulk and regenerates the state to the next PCET cycle. (E) Free energy diagram of the redox-driven proton pumping steps. The values are based on the present calculations and experimental data (see SI Appendix, Extended Methods).