Intramolecular Proton-Transfer Reactions in a Membrane-Bound Proton Pump:  The Effect of pH on the Peroxy to Ferryl Transition in Cytochrome <i>c</i> Oxidase<sup>,</sup>

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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.

Section: Resultsmentioning

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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“…Time-resolved measurements of the R-to-F transition help to define an order (22,33). Because each reduction of the BNC is associated with proton pumping (14,30,(34)(35)(36), it is assumed that the order of substates is the same in each redox state (5). Because this is a cycle, the starting substate is arbitrary ( Fig.…”

Section: Significancementioning

confidence: 99%

“…E286 has a high proton affinity, with an operational pK a for CcO turnover of 9.4 (35). The choice taken here is to have the reduction of the BNC before the reprotonation of E286.…”

Section: Significancementioning

confidence: 99%

Characterizing the proton loading site in cytochromecoxidase

Gunner

2014

Cytochrome c oxidase (CcO) uses the energy released by reduction of O 2 to H 2 O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a 3 , Cu B , Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a 3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.MCCE | bioenergetics | pK a | proton transfer

“…ytochrome c oxidase (CcO) captures the energy from the reduction of oxygen to drive the translocation of protons across the inner mitochondrial (or bacterial) membrane (1)(2)(3)(4)(5)(6)(7)(8). The resulting electrochemical gradient powers the production of ATP, the energy source of the cell.…”

mentioning

confidence: 99%

Kinetic gating of the proton pump in cytochrome c oxidase

Kim

Wikström

Hummer

2009

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.bioenergetics ͉ biological machines ͉ kinetic master equation ͉ respiration C ytochrome c oxidase (CcO) captures the energy from the reduction of oxygen to drive the translocation of protons across the inner mitochondrial (or bacterial) membrane (1-8). The resulting electrochemical gradient powers the production of ATP, the energy source of the cell. CcO takes up four electrons from the outside (P side) of the membrane and four protons from the inside (N side) for the reduction of one dioxygen molecule. Four additional protons are translocated across the membrane from the N side to the P side. Crystal structures of the enzyme from various organisms (3, 4, 9, 10) indicate the electron and proton pathways, formed by conserved residues and cofactors. Extensive experimental and theoretical studies have provided valuable information about these pathways, the reaction sequence, and energetic aspects of catalysis (5,7,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, some key aspects of the fundamental mechanisms governing the coupling between the chemical reaction and vectorial proton translocation are still unclear.Here, the objective is to explain how vectorial proton translocation is accomplished in CcO, with respect to both the driving forces (i.e., proton and electron affinities expressed as pK a values and midpoint redox potentials, respectively, and Coulombic couplings) and the kinetic control (i.e., the ''gating'' effects that result from a dependence of kinetic barriers on the microscopic states). Specifically, we aim to construct ...