Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects

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Understanding the nature of energy transduction in life processes requires a quantitative description of the energetics of the conversion of ATP to ADP by ATPases. Previous attempts to do so have provided an interesting insight but could not account for the rotary mechanism by a nonphenomenological structure/energy description. In particular it has been very challenging to account for the observations of the 80°and 40°rotational substates, without any prior information about such states in the simulation procedure. Here we use a coarse-grained model of F1-ATPase and generate, without the adjustment of phenomenological parameters, a structure-based free energy landscape that reproduces the energetics of the mechanochemical process. It is found that the landscape along the relevant rotary path is determined by the electrostatic free energy and not by steric effects. Furthermore, the generated surface and the corresponding Langevin dynamics simulations identify a hidden conformational barrier that provides a new fundamental interpretation of the catalytic dwell and illuminate the nature of the energy conversion process.bioenergetics | chemical-conformational coupling | molecular motors | coarse grain model U nderstanding the energetics of the biological conversion of ATP to ADP is crucial for elucidating the nature of energy transduction in life processes and also for practical understanding of the action of molecular motors (1, 2). At present it seems that the detailed nature of this biological energy conversion process has remained a major puzzle (1-7). For example, despite the fact that the structures of several ATPases have been elucidated (e.g., refs. 4 and 8) and recent advances in detailed elucidation of some key steps have been made (9-11), it is not completely clear at what stage in the reaction energy is actually released and, in turn, what is the nature of the energy conversion process. Some workers have suggested that the major source of the energy release is the conversion of the free energy of ATP binding into elastic strain, which is subsequently released by a coordinated and tightly coupled conformational mechanism (6). It was also postulated that the energy conversion involves an inertial energy release (12), whereas our works (13, 14) have suggested that the energetics is associated with the electrostatic work, and the origin of the free energy change involves a significant contribution from the charge separation in solution outside the ATPase active sites. Furthermore, biochemical and structural studies have provided a molecular model for the action of ATPases (1-4), and remarkable phenomenological analyses (6, 7) indicated what are the conditions for effective action. However, we still do not have a clear structure function correlation that involves both the chemistry and the conformational coupling. The challenge became even more exciting in view of the remarkable progress in single-molecule studies that directly visualized the γ-subunit rotation in a unidirectional way, while revealing several...

“…We also note that, although we are dealing with a nonequilibrium conversion of t w to d w , the individual steps depicted in Fig. 3 satisfy quasi-equilibrium as discussed in (33).…”

Section: Discussionmentioning

confidence: 99%

Electrostatic origin of the mechanochemical rotary mechanism and the catalytic dwell of F1-ATPase

Mukherjee

Warshel

2011

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152

“…Proton transfer from the Glu to the catalytic site would take place only from one of these configurations. The observed apparent pK a would then reflect the equilibrium constant of the two configurations and their pK a values (50,52). Any changes in the observed pK a as a result of mutations-for example, around the 139 site-were explained in terms of changes in the energy profile for proton transfer through the D pathway and changes in the equilibrium constant for the two configurations of Glu-286.…”

Section: Discussionmentioning

confidence: 99%

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Johansson

Högbom

Carlsson

et al. 2013

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Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ∼5,000. Here, we show that proton uptake at full speed (∼10 4 s −1 ) can be restored in the Asp-132–Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139–Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn 2+ addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn 2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139–Thr/Asp-132–Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

“…This site, often referred to as the "proton-loading site" (PLS), would initially become protonated specifically from the n side (but not the p side) and then release its proton to the p side (but not the n side) (19)(20)(21)(22)(23)(24)(25)(26). The identity of the PLS of the heme-copper oxidases is not known.…”

mentioning

confidence: 99%

Mutation of a single residue in the ba ₃ oxidase specifically impairs protonation of the pump site

Ballmoos

Gonska

Lachmann

et al. 2015

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The ba 3 -type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O 2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba 3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O 2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba 3 cytochrome c oxidase.cytochrome c oxidase | membrane protein | respiration | cytochrome aa 3 | electron transfer