| 1. |
- Faxén, Kristina, 1957-
(författare)
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Active Transport of Ions across Biomembranes : A Kinetic Study of Cytochrome c Oxidase Reconstituted into Phospholipid Vesicles
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ion transport across membranes is of uttermost importance for us. It is the foundation for signaling of various kinds e.g. in the nerve-system. Furthermore, energy from photosynthesis and metabolism is conserved in electrochemical gradients across membranes, maintained by ion pumps. In this thesis I discuss mechanisms of how protons and other ions are translocated across biomembranes against their concentration gradients. I have studied one specific proton pump, cytochrome c oxidase (CytcO) and in the summary I also compare CytcO with two other pumps for which a wealth of structural and functional information has recently been obtained. The data in the articles presented in this thesis support a model were proton pumping can be achieved without simultaneous oxidation of heme a or electron transfer (paper I); where a proton is transferred to the catalytic site before the pump site is protonated (paper IV); and where proton release is preceded by a conformational change (paper II). These observations could be explained by a model involving a conformational change of the pump element, recently proposed from our laboratory1. Furthermore the results from the papers in this thesis show that proton uptake precedes proton release in D2O (paper II). The kinetics of electron transfers linked to proton pumping is solely determined by the pH on the N-side of the membrane (paper III). Finally Zn2+ added on the P-side of the membrane inhibits a specific reaction step (paper IV). In the three pumps described here conformational changes, modulating ion affinities, and the opening and closing of gates, seem to be involved in driving the ions across the membrane. 1. Brzezinski, P. & Larsson, G. (2003) Biochim. Biophys. Acta 1605, 1-13.
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| 2. |
- Moe, Agnes, 1990-
(författare)
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Role of respiratory supercomplexes : Electronic connection between complexes III and IV
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the final step of cellular respiration, electrons are transferred through the respiratory chain to reduce molecular oxygen to water. The energy released in this chain is used to maintain a proton electrochemical gradient across the cell membrane, which is used, for example, by the ATP synthase to produce ATP. The enzyme complexes of the respiratory chain are known to organize in supramolecular assemblies, so-called respiratory supercomplexes.In this work we investigated the functional significance of respiratory supercomplexes consisting of complexes III and IV in mitochondria. By combining structural and kinetic studies we showed that at the commonly assumed "physiological" ionic strength of 150 mM monovalent salt, the water-soluble cyt. c associates with the negatively charged surface of III2-IV1-2 supercomplexes in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. The data showed that one cyt. c diffuses in 2D, between complexes III and IV, indicating a kinetic advantage of forming supercomplexes. These studies also showed different relative orientation of the individual complexes in the supercomplexes from the two yeast species, indicating that 2D diffusion is a general mechanism, not limited to a specific relative orientation of complexes III and IV. More recent data in the literature indicate that a more realistic mimic of intracellular conditions is a monovalent salt concentration of 20 mM. We showed that under these conditions two cyt. c molecules bind simultaneously to the supercomplex. This result further supports a kinetic advantage of forming supercomplexes.We also determined the cryo-EM structure of the obligate III2-IV2 supercomplex from the Gram-positive bacterium Corynebacterium glutamicum. The structure revealed an electronic connection between complexes III and IV by a di-heme cyt. cc subunit. The structure also showed that complexes III and IV are structurally intertwined and strongly connected with unique features conserved in the phylum actinobacteria.
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| 3. |
- Uzdavinys, Povilas, 1985-
(författare)
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Establishing the molecular mechanism of sodium/proton exchangers
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na+/H+ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes.The goal of my thesis is to establish the molecular basis of ion exchange in Na+/H+ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology.In my thesis, I will first present an overview of Na+/H+ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na+/H+ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na+/H+ exchanger (NHA2) in an isolated liposome reconstitution system.
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| 4. |
- von Ballmoos, Christoph, et al.
(författare)
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Proton transfer in ba(3) cytochrome c oxidase from Thermus thermophilus
- 2012
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1817:4, s. 650-657
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Forskningsöversikt (refereegranskat)abstract
- The respiratory heme-copper oxidases catalyze reduction of O-2 to H2O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa(3)-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the,catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba(3) oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H+/electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A- and the B-class ba3 oxidases with a focus on mechanisms of proton transfer and pumping. This article is part of a Special Issue entitled: Respiratory Oxidases.
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| 5. |
- Faxén, Kristina, et al.
(författare)
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The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase.
- 2007
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1767:5, s. 381-386
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).
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| 6. |
- Berg, Johan, 1986-
(författare)
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Proton transfer across and along biological membranes
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
(författare)
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Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry
- 2020
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1861:2
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Tidskriftsartikel (refereegranskat)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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| 8. |
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| 9. |
- Björck, Markus L., et al.
(författare)
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Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In cytochrome c oxidase (CytcO) reduction of O2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than throughthe D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D(Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO withO2. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and pumping.
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| 10. |
- Björck, Markus, 1987-
(författare)
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Regulation of proton-coupled electron transfer in cytochrome c oxidase : The role of membrane potential, proton pathways and ATP
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cytochrome c oxidase (CytcO) is the final electron acceptor of the respiratory chain. In this chain a current of electrons, derived from degradation of nutrients, along with protons, are used to reduce oxygen to water. The reaction is exergonic and the excess energy is used to pump protons across the membrane. This proton-coupled electron transfer is regulated, for example, by the membrane potential, the composition of the membrane and the ATP/ADP concentrations. Here, we have investigated the mechanism of this regulation. Specifically, we investigated ligand binding to CytcO in mitochondria, which provides mechanistic information about CytcO in its native environment. In addition to CytcO, a water soluble protein, flavohemoglobin (yHb) was found to bind CO and we found that it is localized in the intermembrane space (IMS). We also extracted CytcO from mitochondria without detergent using the styrene maleic acid (SMA) co-polymer. We could show that the SMA-extracted CytcO behaved similarly in its reaction with O2 and CO as CytcO in mitochondria.In mitochondria and bacterial membranes CytcO transports charges against a transmembrane electrochemical gradient. We induced a membrane potential across sub-mitochondrial particles (SMPs) by addition of ATP and measured single CytcO turnover. Our results indicate that proton transfer, but not electron transfer, across the membrane is affected by the membrane potential.In yeast CytcO subunit Cox13 has been shown to play a role in ATP/ADP binding to regulate activity. We have solved the structure of Cox13 using NMR and identified the residues that constitute the ATP-binding site, which is located at the C-terminus.Finally we showed that the main proton-transfer pathways in yeast CytcO function similarly to their bacterial counterparts and that the proposed H-pathway, absent in bacteria, is not responsible for proton translocation in mitochondrial CytcO from S. cerevisiae.
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