| 1. |
- Ballout, Fouad, et al.
(författare)
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Scanning near-field IR microscopy of proteins in lipid bilayers
- 2011
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 13:48, s. 21432-21436
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Tidskriftsartikel (refereegranskat)abstract
- We use infrared near-field microscopy to chemically map the morphology of biological matrices. The investigated sample is built up from surface-tethered membrane proteins (cytochrome c oxidase) reconstituted in a lipid bilayer. We have carried out infrared near-field measurements in the frequency range between 1600 and 1800 cm(-1). By simultaneously recording the topography and chemical fingerprint of the protein-tethered lipid bilayer with a lateral resolution of 80 nm x 80 nm, we were able to probe locally the chemical signature of this membrane and to provide a local map of its surface morphology.
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| 2. |
- Hugentobler, Katharina Gloria, et al.
(författare)
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Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:19
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Tidskriftsartikel (refereegranskat)abstract
- The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.
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| 3. |
- Krassen, Henning, et al.
(författare)
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In vitro hydrogen production-using energy from the sun
- 2011
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 13:1, s. 47-57
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Tidskriftsartikel (refereegranskat)abstract
- Using solar energy to produce molecular hydrogen is a promising way to supply the civilization with clean energy. Nature provides the key components to collect solar energy as well as to reduce protons, scientists have developed mimics of these enzymatic centers and also found new ways to catalyze the same reactions. This perspective article surveys the different components and in particular the various coupling possibilities of a light sensitizer and catalyst. Pros and cons are discussed.
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| 4. |
- Krassen, Henning, et al.
(författare)
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Tailor-Made Modification of a Gold Surface for the Chemical Binding of a High-Activity [FeFe] Hydrogenase
- 2011
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Ingår i: European Journal of Inorganic Chemistry. - : Wiley. - 1434-1948 .- 1099-1948 .- 1099-0682. ; :7, s. 1138-1146
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogenases are iron-sulfur proteins that catalyze hydrogen turnover in a wide range of microorganisms. Three different classes have been described, and among these [FeFe] hydrogenases are the most active in H-2 evolution. Hydrogenases are redox enzymes that have been shown to exchange electrons with graphite and modified noble metal electrodes. Making use of the latter, diffusible electron carriers are required to enable redox catalysis, as proteins do not specifically bind to the electrode surface. Diffusion-limited electron transfer can be replaced by electron injection into immobilized hydrogenase by binding the redox mediator to the electrode surface. Here, we present the synthesis and spectroelectrochemical characterization of 1-( 10-mercaptodecyl)-1'-benzyl-4,4'-bipyridinium dibromide (MBBP), which is used as redox-active linker. CrHydA1, the high-activity [FeFe] hydrogenase from Chlamydomonas reinhardtii, is immobilized on the linker-modified gold electrode. Each surface modification step is controlled in situ by surface-enhanced infrared absorption spectroscopy (SEIRAS). Functionality of the electrode-protein hybrid is demonstrated by recording the linker-supported current. The specific catalytic rate of hydrogen evolution by CrHydA1 (2.9 mu molH(2)min(-1)mg(-1) hydrogenase) promises a valuable approach for further optimization of this novel bioelectrical interface.
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| 5. |
- Meszaros, Livia S., et al.
(författare)
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Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase
- 2020
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Ingår i: Chemical Science. - : ROYAL SOC CHEMISTRY. - 2041-6520 .- 2041-6539. ; 11:18, s. 4608-4617
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogenases are among the fastest H-2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species.
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| 6. |
- Nordlund, Gustav, 1980-
(författare)
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Membrane-mimetic systems : Novel methods and results from studies of respiratory enzymes
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The processes localized to biological membranes are of great interest, both from a scientific and pharmaceutical point of view. Understanding aspects such as the detailed mechanism and regulation of these processes requires investigation of the structure and function of the membrane-bound proteins in which they take place. The study of these processes is often complicated by the need to create in vitro systems that mimic the environment in which these proteins are normally found in vivo. This thesis describes some of the methods available for membrane-protein studies in membrane-mimetic systems, as well as our work aimed at developing such systems. Furthermore, results from studies using these systems are described.In the first two studies, described in Papers I & II, we investigated the use of silica particle-supported lipid bilayers, both for membrane-protein studies and as possible drug-delivery vehicles. Successful reconstitution of a multisubunit proton-pump, cytochrome c oxidase is described and characterized. Initial attempts to develop drug-delivery systems with two different targeting peptides are also described in the thesis.The second part of this thesis revolves around our work with membraneprotein dependent pathways. Results from studies of systems where the proton- pump bo3 oxidase and ATP synthase work in concert are described. The results show a surprising lipid-composition dependence for the coupled bo3- ATP-synthase activity (Paper III).Finally, a new system utilizing synaptic vesicle-fusion proteins for coreconstitution of membrane proteins is described, showing successful coreconstitution of a small respiratory chain, delivery of soluble proteins to preformed liposomes and reconstitution of ATP synthase in native membranes (Paper IV).
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| 7. |
- Schubert, Luiz, et al.
(författare)
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Proton Release Reactions in the Inward H+ Pump NsXeR
- 2023
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Ingår i: Journal of Physical Chemistry B. - 1520-6106 .- 1520-5207. ; 127:39, s. 8358-8369
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Tidskriftsartikel (refereegranskat)abstract
- Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.
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| 8. |
- Vilhjálmsdóttir, Jóhanna, 1982-
(författare)
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Choreography of a proton pump : Studies of charge-transfer reactions in cytochrome c oxidase
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the last step of cellular respiration, electrons from metabolites are transferred to molecular oxygen, mediated by the enzyme complexes of the respiratory chain. Some of these enzyme complexes couple these redox reactions to formation of an electrochemical proton gradient across the cell membrane. The proton gradient is used e.g. by ATP synthase to drive synthesis of ATP. The terminal enzyme complex in the respiratory chain, cytochrome c oxidase (CytcO), catalyses reduction of O2 to water. In this process it contributes to maintaining the electrochemical proton gradient by two separate mechanisms: (i) by uptake of electrons and protons from the opposite sides of the membrane (for O2 reduction to water). (ii) by proton pumping across the membrane. Protons used in the O2 reduction, as well as protons that are pumped, are taken up through two different proton-uptake pathways, the D and the K pathways. In addition, a third proton-transfer pathway has been suggested for the mitochondrial CytcOs, namely the H pathway. So far, the molecular mechanism by which CytcO pumps protons has not been determined. In this work we have studied proton- and electron-transfer reactions in aa3-type CytcOs, with the aim of understanding the functional design of the proton-pumping machinery in CytcO. First, we studied structural variants of CytcO from the bacterium Rhodobacter (R.) sphaeroides, where an amino-acid at position 425, previously shown to undergo redox-induced conformational changes, was substituted. The results point to a link between redox-induced structural changes and intramolecular proton-transfer rates through the D pathway. Second, we studied the electron distribution in the “activated” oxidized (OH) state of CytcO, by using an electrostatic complex of CytcO and cytochrome c. We also investigated electron-transfer reactions linked to proton pumping in structural variants of CytcO from R. sphaeroides and the yeast Saccharomyces (S.) cerevisiae, with mutations in the proposed D and H proton-uptake pathways. The data indicate that the S. cerevisiae mitochondrial CytcO uses the D pathway for proton uptake and pumping as the R. sphaeroides CytcO. Lastly, we studied reactions linked to proton uptake and pumping in structural variants of CytcO from R. sphaeroides with alterations in both proton-uptake pathways. The data elucidated the mechanism of proton transfer and gating in CytcO.
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