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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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O2 probability density maps obtained from all-atom MD simulations.A) The probability density contours at 0.00015 Å−3 are depicted as a grey surface. The X-ray structure is represented as a ribbon with subunit I colored in green and subunit II in cyan. The hemes are depicted as green sticks. The yellow, light blue and green spheres represent the Cu, Fe (from the heme groups) and the Mg atoms, respectively. The residues forming the putative O2 channel inferred from the X-ray structure (according to [13]) are colored in orange and the residues forming the constriction point in this channel are highlighted in magenta. B) Zoom image of the putative O2 channel inferred from the X-ray structure pressurized with xenon [13]. The red arrow identifies the two possible entrance points for this O2 channel.
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pcbi-1004010-g002: O2 probability density maps obtained from all-atom MD simulations.A) The probability density contours at 0.00015 Å−3 are depicted as a grey surface. The X-ray structure is represented as a ribbon with subunit I colored in green and subunit II in cyan. The hemes are depicted as green sticks. The yellow, light blue and green spheres represent the Cu, Fe (from the heme groups) and the Mg atoms, respectively. The residues forming the putative O2 channel inferred from the X-ray structure (according to [13]) are colored in orange and the residues forming the constriction point in this channel are highlighted in magenta. B) Zoom image of the putative O2 channel inferred from the X-ray structure pressurized with xenon [13]. The red arrow identifies the two possible entrance points for this O2 channel.

Mentions: Furthermore, during the simulation time, some O2 molecules move from the membrane into the protein. The number of O2 inside Ccox increased slowly during the first 30 ns of simulation until it stabilizes at ∼8 molecules on average (see S9 Figure in S1 Text). In general, before entering Ccox, the O2 molecules explore the protein's surface and bind briefly to the cavities and niches formed mainly by hydrophobic residues. However, after 100 ns, none of the internalized O2 molecules was able to reach the BNC in any replicate. Nonetheless, and in order to determine which regions of the protein are more populated by the O2 molecules during our simulations, we calculated the O2 probability density maps [59] over the 100 ns (for all five replicates) and the results are depicted in Fig. 2.


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)

O2 probability density maps obtained from all-atom MD simulations.A) The probability density contours at 0.00015 Å−3 are depicted as a grey surface. The X-ray structure is represented as a ribbon with subunit I colored in green and subunit II in cyan. The hemes are depicted as green sticks. The yellow, light blue and green spheres represent the Cu, Fe (from the heme groups) and the Mg atoms, respectively. The residues forming the putative O2 channel inferred from the X-ray structure (according to [13]) are colored in orange and the residues forming the constriction point in this channel are highlighted in magenta. B) Zoom image of the putative O2 channel inferred from the X-ray structure pressurized with xenon [13]. The red arrow identifies the two possible entrance points for this O2 channel.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256069&req=5

pcbi-1004010-g002: O2 probability density maps obtained from all-atom MD simulations.A) The probability density contours at 0.00015 Å−3 are depicted as a grey surface. The X-ray structure is represented as a ribbon with subunit I colored in green and subunit II in cyan. The hemes are depicted as green sticks. The yellow, light blue and green spheres represent the Cu, Fe (from the heme groups) and the Mg atoms, respectively. The residues forming the putative O2 channel inferred from the X-ray structure (according to [13]) are colored in orange and the residues forming the constriction point in this channel are highlighted in magenta. B) Zoom image of the putative O2 channel inferred from the X-ray structure pressurized with xenon [13]. The red arrow identifies the two possible entrance points for this O2 channel.
Mentions: Furthermore, during the simulation time, some O2 molecules move from the membrane into the protein. The number of O2 inside Ccox increased slowly during the first 30 ns of simulation until it stabilizes at ∼8 molecules on average (see S9 Figure in S1 Text). In general, before entering Ccox, the O2 molecules explore the protein's surface and bind briefly to the cavities and niches formed mainly by hydrophobic residues. However, after 100 ns, none of the internalized O2 molecules was able to reach the BNC in any replicate. Nonetheless, and in order to determine which regions of the protein are more populated by the O2 molecules during our simulations, we calculated the O2 probability density maps [59] over the 100 ns (for all five replicates) and the results are depicted in Fig. 2.

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