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. 2009 Mar 4;96(5):1721-32.
doi: 10.1016/j.bpj.2008.11.052.

Association of Cytochrome C With Membrane-Bound Cytochrome C Oxidase Proceeds Parallel to the Membrane Rather Than in Bulk Solution

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Association of Cytochrome C With Membrane-Bound Cytochrome C Oxidase Proceeds Parallel to the Membrane Rather Than in Bulk Solution

Alexander Spaar et al. Biophys J. .
Free PMC article

Abstract

Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c(552), and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions.

Figures

Figure 1
Figure 1
Electrostatic field of the DPPC membrane without COX. (A) Isosurfaces at +1 kT/e (blue) and −1 kT/e (red). Note the three-fold periodicity along the x and y dimensions that results from the assembly of the lipid patch from eight identical membrane patches. The central box was equilibrated around COX. For calculation of this electrostatic potential map, the COX charges were switched off. (B) The same isosurfaces (white and gray, respectively) and the phosphorus (red) and nitrogen (blue) atoms of the DPPC headgroups, demonstrating the inhomogeneous charge distribution.
Figure 2
Figure 2
Free-energy landscapes and optimal association pathways for cyt c552 (AC) and cyt ch (DF) with COX. (A, B, D, and E) Top (A and D) and side (B and E) views of free-energy landscapes as isosurfaces at 33% of the corresponding free-energy minimum. Shown are associations of cytochrome c with fully solvated COX (blue), COX embedded in an uncharged membrane (green), and COX embedded in a polar membrane (orange). (C and F) Optimal association pathways and encounter complex conformations, shown as spheres located on the center of mass and as ribbons, respectively. Colors are the same as for isosurfaces. The red ribbon represents cytochrome c in the modeled bound conformation.
Figure 3
Figure 3
Free-energy landscape maps for the energetically most favorable conformations of cyt c552 (A and B) and cyt ch (C and D) with COX. Maps for positional and orientational coordinates for the association of cytochrome c with fully solvated COX (left), COX embedded in a membrane without lipid charges (center); and COX embedded in a membrane with full electrostatics (right). Free energy levels range from low (blue) to high (red) (A and C) Translational free-energy landscapes. (B and D) Orientational free-energy landscapes.
Figure 4
Figure 4
Energy profiles along minimal free-energy pathways. Effects of the membrane on the energy profiles are shown for cyt c552 (solid symbols) and cyt ch (open symbols) with COX (200 mM). The energies are displayed with respect to the distance between protein centers for three different association scenarios for cytochrome c: with fully solvated COX, with COX embedded in an uncharged membrane, and with COX embedded in a polar membrane. The color scheme is the same as for Fig. 2. (A) Electrostatic energy, ΔEel; (B) Desolvation energy, ΔEds; (C) Translational/rotational entropy loss, −TΔS. (D) Free energy, ΔG. The profiles for cyt c552 with COX embedded in a polar membrane are mean values with corresponding standard deviations from five simulations.
Figure 5
Figure 5
Density profiles along the z axis. The effects of the membrane on the density profiles for the association of cyt c552 (A) and cyt ch (B) with COX. The profiles are normalized to the total occupancy in the interval z = 0–100 Å, and all occupancies within a radius of 40 Å in the xy direction around the COX center were omitted to exclude the immediate effect of COX.
Figure 6
Figure 6
Free-energy profiles along minimal free-energy pathways (A) and computed association rates (B). The effects are shown of different redox states on (A) the free-energy profile and (B) the association rates for cyt c552 and membrane-embedded COX (with full electrostatics). The plots show the results from simulations of reduced cyt c552 to fully oxidized COX (solid squares) and to COX only oxidized at the CuA center (solid circles), and from simulations of oxidized cyt c552 to fully reduced COX (open circles) and to COX only reduced at the CuA center (open squares).

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