| 1. |
- Lachmann, Peter, 1980-
(author)
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Electron and Proton Transfer in Nitric Oxide Reductase : NO Binding, NO Reduction and no Pumping
- 2009
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Licentiate thesis (other academic/artistic)abstract
- Nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two electronreduction of NO to N2O (2NO + 2H+ + 2e- → N2O + H2O) as part of the process ofdenitrification, the step-wise reduction of nitrate to dinitogen. The NOR-catalyzedreaction is central in the nitrogen cycle, since in this step the N=N double bond isformed. NOR is a deviant heme copper oxidase, located in the cytoplasmic membrane,containing four redox active cofactors. Like cytochrome c oxidase (CcO), NOR canreduce oxygen to water as a side reaction, but in contrast to CcO it does not contributeto the proton motive force that drives the conversion of ADP to ATP by ATP synthase.The active site in the catalytic subunit NorB consists of a non-heme iron FeB and a hemeb3 that are anti-ferromagnetically coupled. Additionally a low-spin heme b in NorB isinvolved in accepting electrons from heme c of NorC, a membrane anchored cytochromec, which is the second subunit of the purified NorBC heterodimer.We have studied the terminal region of the proton entry channel and possible ligands tothe binuclear active site of NOR using the flow-flash technique and could demonstratethat the putative proton channel residues Glu(E)198 and E267 in NorB are essential forproton uptake. We propose that they define the terminal proton channel region close tothe binuclear site. An alanine variant of the fully conserved amino acid residue E202 ofNOR that, according to the model of NOR (47), is located in the vicinity to the active site,is neither essential for catalytic activity nor integrity of the active site.Furthermore, we were able to demonstrate the [NO] dependency of NOR in the reactionbetween fully reduced protein and NO using the flow-flash technique (21, 24). Thebinding of NO to the fully reduced enzyme is clearly concentration dependent,inconsistent with a previously proposed obligatory binding of NO first to FeB before itligates to heme b3, where it, in the first turnover, is reduced by the electrons from theactive site. Further oxidation involves electron transfer from the low-spin hemes, which isaccelerated at lower [NO]. This acceleration at lower substrate concentration is evenlarger at decreased pH. We could demonstrate that substrate inhibition, observed insteady-state measurements, occurs already on oxidizing the fully reduced enzyme,indicating that NO binds to its inhibitory site before electrons can redistribute to theactive site from the low-spin hemes.
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| 2. |
- Näsvik Öjemyr, Linda, et al.
(author)
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Reaction of wild-type and Glu243Asp variant yeast cytochrome c oxidase with O-2
- 2014
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In: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1837:7, s. 1012-1018
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Journal article (peer-reviewed)abstract
- We have studied internal electron transfer during the reaction of Saccharomyces cerevisiae mitochondrial cytochrome c oxidase with dioxygen. Similar absorbance changes were observed with this yeast oxidase as with the previously studied Rhodobacter sphaeroides and bovine mitochondrial oxidases, which suggests that the reaction proceeds along the same trajectory. However, notable differences were observed in rates and electron-transfer equilibrium constants of specific reaction steps, for example the ferryl (F) to oxidized (O) reaction was faster with the yeast (0.4 ms) than with the bovine oxidase (similar to 1 ms) and a larger fraction Cu-A was oxidized with the yeast than with the bovine oxidase in the peroxy (P-R) to F reaction. Furthermore, upon replacement of Glu243, located at the end of the so-called D proton pathway, by Asp the P-R -> F and F -> O reactions were slowed by factors of similar to 3 and similar to 10, respectively, and electron transfer from Cu-A to heme a during the P-R -> F reaction was not observed. These data indicate that during reduction of dioxygen protons are transferred through the D pathway, via Glu243, to the catalytic site in the yeast mitochondrial oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
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| 3. |
- von Ballmoos, Christoph, et al.
(author)
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Mutation of a single residue in the ba(3) oxidase specifically impairs protonation of the pump site
- 2015
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:11, s. 3397-3402
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Journal article (peer-reviewed)abstract
- 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 similar to 65 mu s 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 similar to 1.2 ms was slowed to similar 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.
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| 4. |
- von Ballmoos, Christoph, et al.
(author)
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Timing of Electron and Proton Transfer in the ba(3) Cytochrome c Oxidase from Thermus thermophilus
- 2012
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:22, s. 4507-4517
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Journal article (peer-reviewed)abstract
- Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O-2 to H2O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O-2 reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H+/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H+/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba3 oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a proton-loading site (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.
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| 5. |
- Yanofsky, David J., et al.
(author)
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Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203)
- 2021
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In: eLIFE. - 2050-084X. ; 10
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Journal article (peer-reviewed)abstract
- The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.
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| 6. |
- Berg, Johan, 1986-
(author)
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Proton transfer across and along biological membranes
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Proton-transfer reactions belong to the most prevalent reactions in the biosphere and make life on Earth possible, as they are central to energy conversion. In most known organisms, protons are translocated from one side of a membrane to the other, which generates an electrochemical gradient that drives ATP synthesis. Both the membranes and the proteins that are involved in these processes are vital components of energy-conversion machineries. This thesis presents and discusses proton transfer at surfaces of membranes and proteins, as well as proton translocation across membranes via enzymes.In the first work, we developed a single-enzyme approach to study proton translocation by the proton pump cytochrome bo3 (cyt. bo3). The generated proton gradients were stable as long as substrate (electrons, oxygen) was available. Individual cyt. bo3 could generate proton gradients of ∼2 pH units, which correspond to the measured electrochemical gradient in Escherichia coli cells.When acidic and basic amino acids are in close proximity to each other on a protein surface, their individual Coulomb cages can merge to form a proton antenna that enables fast proton transfer to specific groups. To investigate how the function of a proton pump is affected by structural changes in a proton antenna, close to a proton uptake pathway, we characterized the function and structure of genetic variants of cytochrome c oxidase (CytcO). When a Glu, located about 10 Å from the first residue of the D-pathway, was replaced by a non-protonatable residue (Ala) the proton pumping efficiency decreased by more than half compared to the wild-type enzyme. The proton-uptake kinetics was also altered in this variant.Cardiolipin (CL) is found in membranes where ATP is generated. This phospholipid alters the membrane structure and binds a variety of proteins including all complexes that take part in oxidative phosphorylation. To investigate the role of CL in proton-transfer reactions on the surface of membranes we used fluorescence correlation spectroscopy to study inner mitochondrial membranes from Saccharomyces cerevisiae. The protonation rate at wild-type membranes was about 50% of that measured with membranes prepared from mitochondria lacking CL. The protonation rate on the surface of small unilamellar vesicles (SUVs) decreased by about a factor of three when DOPC-SUVs were supplemented with 20% CL. Furthermore, phosphate buffer titrations with SUVs showed that CL can act as a local proton buffer in a membrane.The respiratory supercomplex factor 1 (Rcf1) has been suggested to facilitate direct electron transfer from the bc1 complex to CytcO by bridging the enzymes and binding cytochrome c (cyt. c) to a flexible domain of Rcf1. We investigated biding of cyt. c to Rcf1 reconstituted into different membrane environments. The apparent KD of the binding between cyt. c and DOPC-liposomes was almost five times lower when Rcf1 was present in the vesicles. Moreover, the apparent KD between cyt. c and liposome reconstituted CytcO was about nine times lower for CytcO isolated from a wild-type strain compared to a Rcf1-lacking strain.
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| 7. |
- Berg, Johan, et al.
(author)
-
Single Proteoliposomes with E.coli Quinol Oxidase : Proton Pumping without Transmembrane Leaks
- 2017
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In: Israel Journal of Chemistry. - : Wiley. - 0021-2148. ; 57:5, s. 437-445
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Journal article (peer-reviewed)abstract
- Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochromebo(3) from E.coli at single-molecule level. Liposomes with reconstituted cytochromebo(3) were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochromebo(3) in the membrane; in approximate to 70% of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a pH (in the presence of the K+ ionophore valinomycin), which was stable over at least approximate to 800s. No rapid changes in pH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E.coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li etal. J. Am. Chem. Soc. (2015) 137, 16055-16063).
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| 8. |
- Berg, Johan, et al.
(author)
-
Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry
- 2020
-
In: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1861:2
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Journal article (peer-reviewed)abstract
- Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O2-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.
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| 9. |
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| 10. |
- Björck, Markus L., et al.
(author)
-
Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential
- 2018
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
-
Journal article (peer-reviewed)abstract
- The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.
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