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Exploring O2 diffusion in A-type cytochrome c oxidases: molecular dynamics simulations uncover two alternative channels towards the binuclear site.

Oliveira AS, Damas JM, Baptista AM, Soares CM - PLoS Comput. Biol. (2014)

Bottom Line: These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient.In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC.Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

View Article: PubMed Central - PubMed

Affiliation: ITQB - Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

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Overview of the O2 lowest free energy pathways obtained from the ILS free energy landscape.For legend details related with the representation of the free energy minima and the pathways connecting them, see Fig. 3C. Heme a3 is represented in grey sticks whereas the grey spheres correspond to the Cu atom from the CuB center and to the Fe atom from heme a3. The residues forming the O2 channels are shown in sticks with a sequence label. A- Lowest free energy pathway and free energy minima for the O2 channel 1. The free energy values of the represented minima (in kJ·mol−1) are: −21.07 (M1), −11.79 (M2), −10.58 (M3), −17.97 (M4), −17.53 (M5), −16.25 (M6), −13.87 (M7), −20.07 (M8), −20.47 (M9), −20.47 (M10) and −7.12 (M11). B- Lowest free energy pathways and minima for the O2 channel 2 and channel 3. For the O2 channel 2, the values (in kJ·mol−1) for the minima are: −13.87 (M12), 0.14 (M13), −22.77 (M19) and −11.03 (M20). The free energy values (in kJ·mol−1) of the minima forming the O2 channel 3 are: −17.24 (M14), −10.46 (M15), −15.81 (M16), −10.40 (M17), −10.18 (M18), −22.77 (M19) and −11.03 (M20).
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pcbi-1004010-g004: Overview of the O2 lowest free energy pathways obtained from the ILS free energy landscape.For legend details related with the representation of the free energy minima and the pathways connecting them, see Fig. 3C. Heme a3 is represented in grey sticks whereas the grey spheres correspond to the Cu atom from the CuB center and to the Fe atom from heme a3. The residues forming the O2 channels are shown in sticks with a sequence label. A- Lowest free energy pathway and free energy minima for the O2 channel 1. The free energy values of the represented minima (in kJ·mol−1) are: −21.07 (M1), −11.79 (M2), −10.58 (M3), −17.97 (M4), −17.53 (M5), −16.25 (M6), −13.87 (M7), −20.07 (M8), −20.47 (M9), −20.47 (M10) and −7.12 (M11). B- Lowest free energy pathways and minima for the O2 channel 2 and channel 3. For the O2 channel 2, the values (in kJ·mol−1) for the minima are: −13.87 (M12), 0.14 (M13), −22.77 (M19) and −11.03 (M20). The free energy values (in kJ·mol−1) of the minima forming the O2 channel 3 are: −17.24 (M14), −10.46 (M15), −15.81 (M16), −10.40 (M17), −10.18 (M18), −22.77 (M19) and −11.03 (M20).

Mentions: The O2 channel 1 approaches the BNC from the subunit I side and corresponds to the channel inferred from the X-ray data (for R. sphaeroides[13] or for bovine [7]). This pathway has two entry points that are fused together in a free energy minimum located in the constriction point just before the BNC (M10 in Fig. 4A). The free energy profile for this pathway (Fig. 4A) is characterized by a very high permeation free energy barrier in the constriction point (associated to the bulky side chain of W172I) and three deep local minima (M8, M9 and M10 in Fig. 4A) located between W172I, F282I and E286I for M10, L243I, F282I and L283I for M8, and I250I, V194I and F108I for M9. The local minima observed just below the Ccox surface (M1 and M6) can probably act as scavengers for the O2 freely diffusing in the membrane and help to create an O2 reservoir inside the protein. In this pathway, W172I and E286I seem to act as the gateway residues that control O2 access to the catalytic site. Nevertheless, the passage from the constriction point to the BNC (moving from M10 to M11 in Fig. 4A) implies the overcoming of a very high free energy barrier of 39.5 kJ·mol−1 (see Fig. 4A and Fig. 5A), which makes O2 diffusion via this pathway very slow and difficult.


Exploring O2 diffusion in A-type cytochrome c oxidases: molecular dynamics simulations uncover two alternative channels towards the binuclear site.

Oliveira AS, Damas JM, Baptista AM, Soares CM - PLoS Comput. Biol. (2014)

Overview of the O2 lowest free energy pathways obtained from the ILS free energy landscape.For legend details related with the representation of the free energy minima and the pathways connecting them, see Fig. 3C. Heme a3 is represented in grey sticks whereas the grey spheres correspond to the Cu atom from the CuB center and to the Fe atom from heme a3. The residues forming the O2 channels are shown in sticks with a sequence label. A- Lowest free energy pathway and free energy minima for the O2 channel 1. The free energy values of the represented minima (in kJ·mol−1) are: −21.07 (M1), −11.79 (M2), −10.58 (M3), −17.97 (M4), −17.53 (M5), −16.25 (M6), −13.87 (M7), −20.07 (M8), −20.47 (M9), −20.47 (M10) and −7.12 (M11). B- Lowest free energy pathways and minima for the O2 channel 2 and channel 3. For the O2 channel 2, the values (in kJ·mol−1) for the minima are: −13.87 (M12), 0.14 (M13), −22.77 (M19) and −11.03 (M20). The free energy values (in kJ·mol−1) of the minima forming the O2 channel 3 are: −17.24 (M14), −10.46 (M15), −15.81 (M16), −10.40 (M17), −10.18 (M18), −22.77 (M19) and −11.03 (M20).
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pcbi-1004010-g004: Overview of the O2 lowest free energy pathways obtained from the ILS free energy landscape.For legend details related with the representation of the free energy minima and the pathways connecting them, see Fig. 3C. Heme a3 is represented in grey sticks whereas the grey spheres correspond to the Cu atom from the CuB center and to the Fe atom from heme a3. The residues forming the O2 channels are shown in sticks with a sequence label. A- Lowest free energy pathway and free energy minima for the O2 channel 1. The free energy values of the represented minima (in kJ·mol−1) are: −21.07 (M1), −11.79 (M2), −10.58 (M3), −17.97 (M4), −17.53 (M5), −16.25 (M6), −13.87 (M7), −20.07 (M8), −20.47 (M9), −20.47 (M10) and −7.12 (M11). B- Lowest free energy pathways and minima for the O2 channel 2 and channel 3. For the O2 channel 2, the values (in kJ·mol−1) for the minima are: −13.87 (M12), 0.14 (M13), −22.77 (M19) and −11.03 (M20). The free energy values (in kJ·mol−1) of the minima forming the O2 channel 3 are: −17.24 (M14), −10.46 (M15), −15.81 (M16), −10.40 (M17), −10.18 (M18), −22.77 (M19) and −11.03 (M20).
Mentions: The O2 channel 1 approaches the BNC from the subunit I side and corresponds to the channel inferred from the X-ray data (for R. sphaeroides[13] or for bovine [7]). This pathway has two entry points that are fused together in a free energy minimum located in the constriction point just before the BNC (M10 in Fig. 4A). The free energy profile for this pathway (Fig. 4A) is characterized by a very high permeation free energy barrier in the constriction point (associated to the bulky side chain of W172I) and three deep local minima (M8, M9 and M10 in Fig. 4A) located between W172I, F282I and E286I for M10, L243I, F282I and L283I for M8, and I250I, V194I and F108I for M9. The local minima observed just below the Ccox surface (M1 and M6) can probably act as scavengers for the O2 freely diffusing in the membrane and help to create an O2 reservoir inside the protein. In this pathway, W172I and E286I seem to act as the gateway residues that control O2 access to the catalytic site. Nevertheless, the passage from the constriction point to the BNC (moving from M10 to M11 in Fig. 4A) implies the overcoming of a very high free energy barrier of 39.5 kJ·mol−1 (see Fig. 4A and Fig. 5A), which makes O2 diffusion via this pathway very slow and difficult.

Bottom Line: These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient.In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC.Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

View Article: PubMed Central - PubMed

Affiliation: ITQB - Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

Show MeSH
Related in: MedlinePlus