Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Quenneville, Jason; Popović, Dragan M.; Stuchebrukhov, Alexei A.

doi:10.1016/j.bbabio.2005.12.003

Cited by 63 publications

(84 citation statements)

References 64 publications

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“…With this change in pK a,sol the pK′ 7 for His I-334 reported here would move down to 8.3. More recent calculations from the Stuchebrukhov group used a DFT-derived pK a,sol of 9 in aqueous calculations with (104) or without (41) a ferryl-heme a 3 included. The pK a of His I-334 is 2.1 (41) or 5.4 (104) in the OORO state.…”

Section: Resultsmentioning

confidence: 99%

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Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O 2 . Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a 3 and Cu B . If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK a s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu B shifts its pK a from below 7 to above 7 and so picks up a proton when heme a 3 and Cu B are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a 3 all have pK a s above 7 in all redox states, although they have only 1.6-3.5 ∆pK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK a s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu A , heme a, heme a 3 , and Cu B shows ≈2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E m ) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E m of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E m is independent of the BNC redox state.Heme-copper oxidases are the terminal electron acceptors in anaerobic organisms. These transmembrane proteins reduce dioxygen and convert the released chemical energy into an electrochemical gradient, across the eukaryotic mitochondrial membrane or the bacterial cell membrane (1-4). Cytochrome c oxidases are the most prevalent hemecopper oxidases. In this protein 4 electrons, provided by 4 cytochromes c, are used to reduce dioxygen to water. The 4 protons needed to make water are taken from the negative cytoplasmic side of the membrane, adding to the electrochemical gradient. Four additional protons are pumped across the membrane (5). Thus, each dioxygen molecule reduced by cytochrome c oxidase is coupled to the transfer of 8 charges across the membrane.The first step of electron transfer is from cytochrome c to Cu A , a dicopper center in subunit II which extends beyond the membrane on the proton release side of the protein (Figure 1). After receiving the electron, Cu A reduces the 6-coordinate, low-spin, bis-Hi...

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Section: Resultsmentioning

confidence: 99%

“…More recent calculations from the Stuchebrukhov group used a DFT-derived pK a,sol of 9 in aqueous calculations with (104) or without (41) a ferryl-heme a 3 included. The pK a of His I-334 is 2.1 (41) or 5.4 (104) in the OORO state. The latter calculation includes the ferryl-heme a 3 in the reference state.…”

Section: Resultsmentioning

confidence: 99%

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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“…However, it is not known what residue binds the proton or if the same residue is used in each redox state (22)(23)(24)(25). The propionic acids on the A (PRA) or D (PRD) rings of heme a 3 (26) or His A334, which is a ligand to Cu B , have all been proposed to be the PLS (15,24,27,28). The energetics and pathway of proton pumping has been simulated assuming the PRD of heme a 3 is the PLS (26,(29)(30)(31)(32).…”

Section: Significancementioning

confidence: 99%

“…Continuum electrostatic calculations have been used to evaluate the protonation states of all residues and the E m of heme a with the BNC fixed in different ionization states (24,41,42). Analysis of the proton acceptors near the BNC in each transition showed with four electrons added to the BNC, only two protons not four were bound (41,42).…”

Section: Significancementioning

confidence: 99%

Characterizing the proton loading site in cytochromecoxidase

Gunner

2014

Proc. Natl. Acad. Sci. U.S.A.

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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

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“…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.…”

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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

Proc. Natl. Acad. Sci. U.S.A.

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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

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Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Cited by 63 publications

References 64 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Characterizing the proton loading site in cytochromecoxidase

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

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Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Cited by 63 publications

References 64 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Characterizing the proton loading site in cytochromecoxidase

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

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