Water−Hydroxide Exchange Reactions at the Catalytic Site of Heme−Copper Oxidases

Brändén, Magnus; Namslauer, Andreas; Hansson, Örjan; Aasa, Roland; Brzezinski, Peter

doi:10.1021/bi0347407

Cited by 30 publications

(48 citation statements)

References 30 publications

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“…After 2 electrons enter the BNC to form the R (OORR) intermediate, the aquo-cofactor's pK a s are greater than 7 so both remain protonated as calculated previously (33). This result agrees with experiments that show little proton release at pH 7 when CO is photolyzed off the mixed valence complex, initiating backelectron transfer moving from the OORR to OROR state (34). The pK a obtained from the fractional site ionization in Monte Carlo sampling as a function of pH for aquo-heme a 3 is 8.6, in good agreement with the measured pK a of 9 for proton release.…”

Section: Resultssupporting

confidence: 87%

“…Thus, with Cu B reduced, the reduction of heme a, not heme a 3 , is calculated to be coupled to the proton binding into the BNC (Table 1). When the second electron is transferred from heme a into the BNC, there are only small changes in total protonation, in agreement with the experiments photolyzing the CO-heme a 3 bond in the mixed valence OORR state (34). Reduction of all four cofactors moving from the OOOO to RRRR state is coupled to the uptake of 2.5 protons ( Table 1).…”

Section: Resultssupporting

confidence: 85%

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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: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 85%

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

2006

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“…Results from earlier studies have shown that the PCET rate is independent of the composition of the buffer or its concentration (17,21), which indicates that buffer molecules do not act as proton donors or acceptors for the protons released through the K pathway. As the observed PCET rate represents the sum of the release and uptake rates, it is not straightforward to calculate a second-order proton-uptake rate constant.…”

Section: Discussionmentioning

confidence: 97%

“…The proton donor is a water molecule at the catalytic site, which interacts electrostatically with heme a 3 . Upon proton release, the formed hydroxide binds to the oxidized heme a 3 (21). Even though this proton release occurs in the opposite direction to that during turnover, the kinetics of the proton-coupled electron transfer (PCET) reflects both the proton uptake and release rates (the sum of these rates) through the K pathway (15,17).…”

mentioning

confidence: 99%

Functional interactions between membrane-bound transporters and membranes

Öjemyr

Lee

Gennis

et al. 2010

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

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One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated proteinprotein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the twodimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.cytochrome aa3 | electron transfer | energy transduction | membrane protein | respiration

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“…Calculations are also carried out on cytochrome c oxidase. Here the pK a is compared with the pH dependence of the proton release on oxidation of heme a 3 (53). Several of these proteins have positively charged groups in the active site.…”

mentioning

confidence: 99%

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

2006

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The pK a s of ferric aquo-heme and aquo-heme electrochemical midpoints (E m s) at pH 7 in sperm whale myoglobin, Aplysia myoblogin, hemoglobin I, heme oxygenase 1, horseradish peroxidase and cytochrome c oxidase were calculated with Multi-Conformation Continuum Electrostatics (MCCE). The pK a s span 3.3 pH units from 7.6 in heme oxygenase 1 to 10.9 in peroxidase, and the E m s range from -250 mV in peroxidase to 125 mV in Aplysia myoglobin. Proteins with higher in situ ferric aquo-heme pK a s tend to have lower E m s. Both changes arise from the protein stabilizing a positively charged heme. However, compared with values in solution, the protein shifts the aquo-heme E m s more than the pK a s. Thus, the protein has a larger effective dielectric constant for the protonation reaction, showing that electron and proton transfers are coupled to different conformational changes that are captured in the MCCE analysis. The calculations reveal a breakdown in the classical continuum electrostatic analysis of pairwise interactions. Comparisons with DFT calculations show that Coulomb's law overestimates the large unfavorable interactions between the ferric water-heme and positively charged groups facing the heme plane by as much as 60%. If interactions with Cu B in cytochrome c oxidase and Arg 38 in horseradish peroxidase are not corrected, the pK a calculations are in error by as much as 6 pH units. With DFT corrected interactions calculated pK a s and E m s differ from measured values by less than 1 pH unit or 35 mV, respectively. The in situ aquo-heme pK a is important for the function of cytochrome c oxidase since it helps to control the stoichiometry of proton uptake coupled to electron transfer [Song, Michonova-Alexova, and Gunner ( . Heme oxygenase is an essential protein in heme metabolism, using three O 2 s and seven electrons to degrade heme to biliverdin (3). Peroxidases reduce toxic hydrogen peroxide to water and then carry out one electron oxidization of a wide range of substrates (4,5). Cytochrome c oxidase is the terminal electron acceptor in the respiratory chain (6-9). Here Heme a 3 forms a binuclear center with a copper complex (Cu B ) that reduces O 2 to water. The protein uses the released chemical energy to pump protons across the membrane. Other five-coordinate heme proteins carry out NO storage and transport (10), steroid synthesis (11, 12), and O 2 , CO, and NO sensing (13,14).Hemes can carry out many biological functions because the protein, especially the active site residues, modulates the ligand binding specificity and resultant chemistry. The diversity of in situ heme functions highlights important interactions of proteins with their cofactors and substrates. Extensive studies have explored how ligand geometry (15-17) and the protein scaffolding (18) affect in situ heme properties (1). The range of redox free energy and pK a s of protein-bound hemes and their ligands show the importance of electrostatic interactions (19,20). For example, sixcoordinate bis-His-hemes have E m s ranging f...

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Water−Hydroxide Exchange Reactions at the Catalytic Site of Heme−Copper Oxidases

Cited by 30 publications

References 30 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?

Functional interactions between membrane-bound transporters and membranes

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

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Water−Hydroxide Exchange Reactions at the Catalytic Site of Heme−Copper Oxidases

Cited by 30 publications

References 30 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?

Functional interactions between membrane-bound transporters and membranes

Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

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Product

Resources

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Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands