Identification of cardiolipin binding sites on cytochrome c oxidase at the entrance of proton channels

Abstract

The respiratory chain or oxidative phosphorylation system (OxPhos) generates most of the chemical energy (ATP) used by our cells. The cytochrome c oxidase (CcO) is one of three protein complexes of OxPhos building up a proton gradient across the inner mitochondrial membrane, which is ultimately used by the ATP synthase to produce ATP. We present molecular dynamic simulations of CcO in a mimic of the mitochondrial membrane, and identify precise binding sites of cardiolipin (CL, signature phospholipid of mitochondria) on the protein surface. Two of these CL binding sites reveal pathways linking CLs to the entrance of the D and H proton channels across CcO. CLs being able to carry protons our results strongly support an involvement of CLs in the proton delivery machinery to CcO. The ubiquitous nature of CL interactions with the components of the OxPhos suggests that this delivery mechanism might extend to the other respiratory complexes.

Figure 1. Structural characteristics of bovine heart cytochrome c oxidase (CIV).

(A) Structure of CIV with its thirteen subunits (A–M) shown in a cartoon representation with a chain-based color-code for one monomer. The subunit's nomenclature following a roman number code used in others studies is given in Supplementary Information. We kept on the letter code to avoid confusion with the site names. In the bottom view of the dimer structure as found in the X-ray structure CLs are depicted. There are three CL binding sites per monomer (CL1a, CL1b and CL2), with one facing the matrix side (CL2) and two facing the intermembrane space (IMS; CL1a and CL1b). CL3 indicates the location of an additional binding site suggested by photolabeling experiments. (B) Simulation box for CIV system, with the protein shown in green, POPC molecules in gray/white, CLs in red/orange and the aqueous phase in blue. (C) Structure of a cardiolipin molecule in an atomistic (AA) and a coarse-grain (CG) representation.

Figure 2. Cardiolipin (CL) binding sites on cytochrome c oxidase (CIV).

Binding sites are extracted from a 100 μs of CGMD simulation of the complex embedded in a CL/POPC membrane bilayer. (A) From left to right: matrix, membrane (two orientations) and inter membrane space (IMS) views of CIV with the CL densities shown in yellow volume maps at an isovalue corresponding to at least 5 times the bulk density. The protein is shown as shaded grey cylinders with the CL densities projected onto them. (B–H) Detailed description of the CL binding sites I to VII, respectively. The residues are numbered as follows: “chain:residue^sub-site”. For each site, the subunits involved in the interactions with the CLs are depicted as colored cartoon as in Fig. 1. The rest of the protein is shown in a transparent gray cartoon.

Figure 3. Potential of mean force for binding of various lipids to sites I, II and III.

(A) Comparison of CL's binding strength to site I (cyan), II (blue) and III (green). (B) Comparison of binding strength of TGL (yellow), POPC (orange) and CL to site II. Two CLs were tested; double charged (−2 e) and neutral, blue and green curves, respectively. In both panel the relative free energy of the system is expressed as a function of the distance, d_COM, between the center of masse of the lipid headgroup and of the binding site as defined in Fig. 2. The error on the measure (estimated using the Bayesian bootstrapping method) is shown by the shaded area behind the curves.

Figure 4. Residue content of the CL binding sites of cytochrome c oxidase (CIV).

The gray sticks indicate the percentage of each residue type at least once in contact with a CL; e.g. present in the section of the CIV accessible to CLs. The black sticks give for each residue type the percentage of its participation to the CL binding sites.

Figure 5. Location of the co-crystallized phospholipid positions and densities computed with our CGMD simulation.

The experimental positions (upper part) were extracted from the PBD entry 2dyr. The densities (lower part) are extracted from the 100 μs CGMD simulation. Views from the matrix (left) and the IMS (right) sides of CIV are shown. CL: cardiolipin; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; TGL: triglyceride.

Figure 6. H-bond networks leading from the cardiolipin (CL) binding sites to the entrance of D- and H-pathways, A:D91 and A:D407, respectively.

The CL binding sites are shown in stick representation. The yellow surface maps depict the CL densities shown in Fig. 2. A:D91 and A:D407 are shown in large sticks. The residues and water molecules (red spheres) participating to the networks are shown in a ball-and-stick representation and numbered as in Fig. 2. The bottom row shows side and top views of CIV with the CL densities, the residues involved in the sites in blue, the residues involved in the transmembrane section of the proton pathways (red arrows) in orange spheres and the heme molecules in green. The large red spheres position the entrances (A:D91 and A:D407) of the pathways.