Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water and uses the released free energy to pump protons against the transmembrane proton gradient. To better understand the proton-pumping mechanism of the wild-type (WT) CcO, much attention has been given to the mutation of amino acid residues along the proton translocating D-channel that impair, and sometimes decouple, proton pumping from the chemical catalysis. Although their influence has been clearly demonstrated experimentally, the underlying molecular mechanisms of these mutants remain unknown. In this work, we report multiscale reactive molecular dynamics simulations that characterize the free-energy profiles of explicit proton transport through several important D-channel mutants. Our results elucidate the mechanisms by which proton pumping is impaired, thus revealing key kinetic gating features in CcO. In the N139T and N139C mutants, proton back leakage through the D-channel is kinetically favored over proton pumping due to the loss of a kinetic gate in the N139 region. In the N139L mutant, the bulky L139 side chain inhibits timely reprotonation of E286 through the D-channel, which impairs both proton pumping and the chemical reaction. In the S200V/S201V double mutant, the proton affinity of E286 is increased, which slows down both proton pumping and the chemical catalysis. This work thus not only provides insight into the decoupling mechanisms of CcO mutants, but also explains how kinetic gating in the D-channel is imperative to achieving high proton-pumping efficiency in the WT CcO.

Keywords: cytochrome c oxidase; decoupling mutants; multiscale; proton pump; proton transport.

Fig. 1.

(A) Overview of the PT pathways (blue arrows) and ET (red arrow) in CcO, highlighting the heme groups and important residues in licorice color; D132, E286, PRDa₃, and the PLS in yellow; and the BNC Cu_B in orange. (B) Reaction scheme during the P_R → F transition with labeled redox groups and protonatable groups. Arrows show the PT processes involved in the chemical reaction (blue), pumping (black), and the E286 reprotonation (blue). The solid arrows indicate PT events that actually happen. The dashed gray arrows indicate potential proton back-leakage pathways that would decouple pumping from the chemical reaction.

Fig. S1.

(A–D) The structure near residues X139 (where X is the amino acid mutation), D132, and N121 in the (A) WT, (B) N139T, (C) N139C, and (D) N139L mutant of CcO. D132 is shown in green and the excess proton in purple. The N139, T139, C139, L139, and N121 residues and water molecules are shown as sticks. The mutant structures are taken from umbrella-sampling windows that have protonated D132 and are on the minimum free-energy pathway of the 2D-PMFs in Fig. 2. The WT structure is similarly taken from the previous work (27).

Fig. 2.

(A–E) The 2D-PMFs (free-energy surfaces) for PT from D132 to E286 in the D-channel of (A) WT, (B) N139T, (C) N139C, (D) N139L, and (E) S200V/S201V. The PMFs are calculated as a function of the excess proton charge defect CEC and the water density along the PT pathway. The 2D-PMF for WT was calculated for the F state and was published in ref. . The 2D-PMFs for the N139T and N139C mutants were calculated for the F state, whereas those for the N139L and S200V/S201V mutants were calculated for the P_R state (as explained in text). The 1D minimum free-energy pathways are depicted as black curves.

Fig. 3.

The 1D free-energy traces for PT through the D-channel of WT (solid and dashed black curves), N139T (blue curve), N139C (purple curve), N139L (red curve), and S200V/S201V (yellow curve), along the minimum free-energy pathways of the 2D-PMFs in Fig. 2. The free-energy traces for the N139T and N139C mutants are calculated for the F state, whereas those for the N139L and S200V/S201V mutants are calculated in the P_R state. For WT, the free-energy trace of both the P_R and the F states was calculated and published in ref. . Note that the N139T trace ends at ∼31 Å simply because the PT pathway in the D-channel is shorter in this mutant. This is a result of the less-curved pathway through the T139 residue.