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G protein coupled receptors (GPCRs) constitute the largest family of cell-surface receptors in mammals and are key players in signal transduction. By responding to a plethora of extracellular stimuli ranging from photons to amines to fatty acids to peptides and proteins, these receptors trigger intracellular signalling cascades and regulate a variety of cellular responses. Approximately 800 genes in humans encode GPCRs which are classified according to sequence conservation into rhodopsin-like, glutamate, adhesion, frizzled/taste2 and secretin receptors. GPCRs share a seven transmembrane domain fold undergoing a conformational change upon ligand binding which is translated to the intracellular surface of the receptor thereby allowing a heterotrimeric G protein to couple. Heterotrimeric G proteins consist of a Ga, Gb and Gg subunit and dissociate into their Ga and Gbg entities upon activation by a GPCR. Subsequently, distinct signalling cascades are triggered by each G protein protomer.
Membrane proteins and GPCRs in particular, are highly important targets in drug design and development as currently approximately 60% of all marketed drugs target membrane proteins. Although these classes of proteins are of high therapeutic interest, our understanding of their mechanism of action and structure remains limited. The first structure of a human GPCR was determined in 2007 and required the development of protein engineering and innovative crystallisation techniques. Since then, approximately 130 GPCR structures of less than 40 individual receptors have been determined providing insights into the structural arrangement of the transmembrane helices, ligand binding pockets and G protein interactions. Combined with spectroscopic methods, these studies allowed a more detailed understanding of the molecular aspects of GPCR activation and signalling. Despite the tremendous advances in GPCR structural biology, certain aspects of GPCR function still remain poorly understood. Due to their size and inherent flexibility, the interaction of protein and peptide ligands with their receptors remains a challenging aspect in the structural characterisation of GPCRs. Moreover, structural information on subtype selectivity of peptide ligands continues to be scarce. To contribute functional and structural information on the molecular mechanisms of peptide interactions with GPCRs, this thesis focused on characterising receptors from the chemoattractant cluster using radioligand binding assays as well as NMR spectroscopy.
The chemoattractant cluster mainly groups the kinin, angiotensin, anaphylatoxin chemotactic complement and apelin receptors according to conserved residues in their ligand binding cavities. All receptors in this cluster bind to peptide ligands deriving from high molecular weight protein precursors upon proteolytic processing. Comparable to the conserved binding pocket of the chemoattractant receptors, the peptide ligands display a certain sequence conservation although they differ strongly in size. The largest ligands used in this thesis are the anaphylatoxins complement 3a and 5a, comprising 77 or 74 residues, respectively. Due to their size and complex fold involving three intramolecular disulphide bonds, solid phase synthesis is impossible, which prompted us to develop a modified cell-free expression system to produce these ligands in tritiated form for subsequent functional characterisation of the complement receptors. To demonstrate the versatility of the developed system, it was applied to another disulphidebond containing peptide ligand, the 21 amino acid endothelin-1. We describe a reliable and multifaceted tool to generate custom labelled peptide ligands for the structural and functional characterisation of GPCRs. The system allows the production of custom radioligands, peptides labelled for NMR studies or with fluorescent amino acids.
Apart from the modulation of GPCR activity by orthosteric ligands, GPCR signalling has long been described to be regulated by allosteric ligands including peptides, small molecules and ions. In this thesis, the influence of sodium ions on the activity state of the chemoattractant cluster receptors and in particular on the apelin, bradykinin 2 and angiotensin II type 1 receptors was examined. In recent high resolution crystal structures an allosteric sodium ion pocket beneath the orthosteric ligand binding cavity was identified and residues contributing to the coordination of sodium ions are conserved throughout the chemoattractant cluster receptors. This allosteric sodium ion coordinated within the transmembrane domain bundle has been described to negatively influence the affinity of agonists but not of antagonists. It was found that sodium ions have distinct influences on the affinity state as well as the available number of binding sites of the chemoattractant receptors. In case of the apelin and bradykinin 2 receptors, sodium ions drastically reduced the number of available binding sites whereas the affinity of peptide ligands to the bradykinin 2 receptors remained constant and the ligand binding affinities to the apelin receptor were completely abolished. In contrast, the angiotensin II type 1 receptor affinity state towards the endogenous peptide ligand angiotensin II is highly dependent on the presence of sodium ions, whereas binding of the synthetic peptide antagonist Sar1-Ile8-angiotensin II remained unaffected by the sodium ion concentration. As differential effects irrespective of the efficacy class but dependent on the amino acid composition of the applied ligands are observed, it can be concluded that electrostatic interactions between charged residues of the peptide ligands and amino acids on the extracellular surface of the receptors are influenced by sodium ions thereby adding another layer of complexity on GPCR signalling.
To elucidate the structure-function relationship of ligand selectivity between the kinin receptors, the structure of desArg10-kallidin (DAK) bound to the bradykinin 1 receptor was determined using solid state NMR (SSNMR) in the course of this thesis. The kinin peptides DAK and bradykinin bind with high affinity and high selectivity to either the bradykinin 1 or bradykinin 2 receptor, respectively. The binding pockets of the receptors are highly conserved and the two peptide ligands only differ in one amino acid at their N- and C-termini whereas the remaining eight amino acids are fully conserved. DAK adopts a U-shaped structure when bound to the bradykinin 1 receptor which resembles a horse shoe-like conformation. Using 2D TEDOR spectroscopy it could furthermore be demonstrated that positively charged residues at the N-terminal part of the peptide engage in ionic interactions with negatively charged amino acids on the extracellular surface of the bradykinin 1 receptor. In contrast, bradykinin displays a distinct b-turn at the C-terminus and an S-shaped conformation of the N-terminal segment when bound to the bradykinin 2 receptor. By using SSNMR to study the binding mode of DAK on the bradykinin 1 receptor we could determine that subtype selectivity between the kinin receptors is conferred by distinct conformational restraints within the peptide ligands and by the formation of specific ionic interaction between charged residues on the peptide and receptor, respectively.
In brief, this thesis contributes structural and functional data on the binding mechanisms and binding mode of different peptide-ligand GPCRs helping to understand subtype selectivity and allosteric modulation of the chemoattractant cluster receptors. In addition, a versatile cell-free expression system was developed that allows the custom synthesis of isotopically labelled peptides containing disulphide bonds for the functional characterisation of GPCRs.
Heme-copper oxidases (HCOs) are the terminal enzymes of the aerobic respiratory chain in the inner mitochondrial membrane or the plasma membrane in many prokaryotes. These multi-subunit membrane protein complexes catalyze the reduction of oxygen to water, coupling this exothermic reaction to the establishment of an electrochemical proton gradient across the membrane in which they are embedded. The energy stored in the electrochemical proton gradient is used e.g. by the FOF1-ATP synthase to generate ATP from ADP and inorganic phosphate. The superfamily of HCOs is phylogenetically classified into three major families: A, B and C. The A-family HCOs, represented by the well-studied aa3-type cytochrome c oxidases (aa3-CcOs), are found in mitochondria and many bacteria. The B-family of HCOs contains a number of bacterial and archaeal oxidases. The C-family comprises only the cbb3-type cytochrome c oxidase (cbb3-CcO) and is most distantly related to the mitochondrial respiratory oxidases.
The four subunit (SU) aa3 cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. It uses electrons from cytochrome c to reduce molecular oxygen to water. Its binuclear active center, residing in SU I, contains hemeÊa3 and CuB, the latter being liganded by three histidine residues. Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity.
To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT CcO was constructed, carrying the gene for SUÊI on a low copy number plasmid. This rec. WT showed, as expected, no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential overproduction of SUÊI due to plasmid coding and the resulting deficiency in metal inserting chaperones might impair the correct insertion of hemeÊa3 and CuB because of a deficiency in metal inserting chaperones. This in turn might lead to differences in side chain orientation and to changes in the water network. However, slight changes might cause an increased accessibility of the active center for hydrogen peroxide, resulting in an increased catalase activity. The availability of chaperones and therefore the proposed structural reasons for the difference was improved by cloning the genes for the two metal inserting chaperones CtaG and Surf1c on the same plasmid together with SUÊI. This new rec. WT CcO showed in fact a reduced catalase activity. Another WT with a deletion in the chromosomal second, non expressing gene of SU I was analysed to prove plasmid coding as the reason for the difference of the ATCC WT and the rec. WT. This strain showed an increased kcat of the catalase activity as well, additionally pointing to a regulatory effect of the non expressed gene for SU I in the chromosome. To fathom the structural difference of the increased catalase activity, differential scanning calorimetry was used, but no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging, the thermal stability of the rec. WT CcO declined faster than that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.
To characterize the catalase reaction, several known inhibitors were used to probe the contribution of the different metal cofactors in the catalase reaction. In addition variants in aromatic amino acids near the active center were constructed to conclude on a possible reaction mechanism of the catalase activity of CcO. These variants in combination with the wild type forms were analysed for radical signals by EPR-spectroscopy. A radical relevant for the catalase reaction of CcO was found in the F-intermediate of all variants and all wild type forms. This narrow 12 G radical signal was assigned to a porphyrine radical probably involved in the catalase reaction of CcO. Moreover, gas chromatography-mass spectrometry measurements were used to analyse isotopically labelled oxygen produced in the catalase reaction.
As a result of these experiments, a reaction cycle of the catalase activity of CcO is postulated and the structural difference between the ATCC and rec. WT CcO is outlined. The catalase activity appears to be a true catalase activity and not a "pseudocatalase" activity.
This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work.
First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits.
Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO.
Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group.
In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.
Cytochrome c oxidase (CcO), also called Complex IV of the aerobic respiratory chain, is located in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. The redox energy of dioxygen reduction is used to translocate protons across the membrane resulting in an electrochemical proton gradient. The generated proton gradient is exploited by the adenosine-5’-triphosphate synthase. In this work, bacterial four-subunit aa3-Type CcO from Paracoccus denitrificans (ATCC 13543, 4 SU-wt ATCC CcO) was used for analyses. 1) The recombinant homologously produced 4 SU-wt CcO (4 SU-wt rec CcO) was functionally compared with the native 4 SU-wt ATCC CcO. The 4 SU-wt rec CcO showed functional deficiencies as determined by UV-vis spectroscopy and electron paramagnetic resonance (EPR) studies. Total X-ray Reflection Fluorescence measurements show in both wild type CcOs the same ratio of the redoxactive Fe and Cu (2 Fe : 3 Cu) indicating full complement of the functional metals. If CcO contains only subunit I and II, it loses its functional integrity during continuous turnover activity. The importance of subunit III for integrity of CcO was demonstrated using 2 SU-wt rec CcO. Crystallisation trials of suicide inactivated 2 SU-wt rec CcOs have been ineffective using standard crystallisation conditions. Crystals of active 2 SU-wt rec CcO (positive control) have been obtained under these conditions and this result indicates possible structural changes in suicide inactivated 2 SU-wt rec CcO. The structure of active 2 SU-wt rec CcO was determined to 2.25 Å resolution. 2) Terminal oxidases require four electrons for the cleavage of the dioxygen bond (O=O). In general, the catalytic cycle of CcO is described by the electron input and thus by the different redox states of the metal centres: the O, E, R, P and F state. The two-electron reduced R intermediate is able to donate four electrons for dioxygen reduction forming the P state. The P intermediate is an oxoferryl state implying the lack of an electron for the R -> P transition, because the metal centres can only provide three electrons (Fe+II forms Fe+IV and Cu+II forms Cu+I). The P state, where the dioxygen bond is already broken, shows an oxoferryl state (FeIV=O2-) and a nearby tyrosine is proposed to form a tyrosyl radical representing the donor of the missing electron. H2O2-induced artificial intermediates provide the opportunity to investigated different catalytic intermediates in detail. Mixing equimolar amounts of H2O2 to CcO in the O state induces the "two-electron" reduced PH state at high pH and the electronically equal "two-electron" reduced F• H state at low pH. The addition of an excess amount of H2O2 leads to the three-electron reduced FH state. Functional studies using the 4 SU-wt ATCC CcO have demonstrated a bound peroxide (O- - O-) intermediate during the catalytic cycle. Using EPR it was previously shown that Y167 hosts a radical species in PH/F• H state which suggests that Y167 could provide this "missing electron". While X-ray structural models of CcO and Fourier-transformed infrared (FTIR) measurements of oxygenated ("pulsed") 4 SU-wt ATCC CcO suggest a bound peroxide in the O state, UV-vis and EPR spectroscopic studies indicate that other intermediates may also contain such peroxide species. Equimolar and excess amounts of H2O2 induce the PH/F• H and FH states, respectively and catalase treatment of the FH state leads, contrary to the natural direction of the catalytic cycle, to the apparent transition of the FH -> PH/F• H states, which is accompanied by reappearance of an EPR signal from the Y167• radical. The novel PFH/F• FH states are presented here and we postulate that the FH state hosts a superoxide (or peroxide) adduct at CuB in the binuclear site. In addition, the novel P10 state is also introduced having a maximum at lambda = 612 nm in the difference absorption spectrum (minus the O state). The P10 state is induced by mixing CcO in the O state with a pH 10 buffer. This pH 10 induced state resembles standard P states such as PCO, PH and PR. However, the P10 state evolves out of the O state without addition of reduction equivalents. Using EPR spectroscopy it was shown that Y167 hosts a radical species in the P10 state such as in the PH state. In summary, all functional data presented here provide evidence for a peroxide bound during the O state. Finally, a new model for the natural catalytic cycle is proposed. If the O state contains a peroxide, it is also likely that the E and R state contain this species. Even the oxoferryl intermediates P and F states may complex a peroxide at CuB in the binuclear site. 3) The amino acid residue Y167, which hosts the radical in the PH/F•H states, is not directly part of the binuclear site of CcO. For identification of the primary electron donor, two tryptophan variants of CcO, W272F and W164F, which are located nearby the binuclear site, were produced. Evidence is provided that W272 is a kinetically fast electron donor for the O2 molecule. The electron is replenished by Y167, or probably by Y280 in the natural cycle. The Y167 radical is detectable by EPR spectroscopy after treatment with equimolar amounts of H2O2 in the active variant W164F, but is absent in the inactive variant W272F. 4) CcO contains two proton conducting pathways, the D- and the K-pathway. Proteoliposomes of the variants H28A and D30N, mutations located at the entrance of the D-pathway, both show the identical proton pumping activity as the 4 SU-wt rec CcO (pumped H+/e- = 1). The variant N113D shows abolished proton pumping (pumped H+/e- = 0), but a relative high cytochrome c oxidation activity (63 %). G196D displays no cytochrome c oxidation and proton pumping activity. Overall, the addition or removal of a negative charge within the D-pathway such as in D124N, N131D, N113D and G196D leads to a decoupled phenotype indicating the high degree of electrostatic coupling in CcO.
Respiration is one of the key processes of energy transduction used by the cell. It consists of two components: electron transfer and ATP production. The electron transfer chain converts the energy released from several biochemical redox reactions into an electrochemical proton gradient across membranes. This stored energy is used as the driving force for the production of ATP by the ATP synthase. The mitochondrial electron transfer chain contains four major protein complexes called complexes I-IV, with counting starting at the lower side of the redox potentials. It has been discussed for a long time how these protein complexes are organized in the membranes. Do they diffuse freely in the membrane? Alternatively, do they form a supercomplex built up of several neighboring complexes? The evidence supporting the free diffusion mode is that both electron transfer intermediates (cytochrome c and quinone) behave as “pool”. However, respiratory supercomplexes have been detected in membranes from bacteria, fungi, yeast, plant and animal during the last decade, and sometimes the respiratory complexes are only stable inside a supercomplex. Therefore, the idea of supercomplex formation has become more popular. The argument that the supercomplex arises from solubilization and is a detergent artifact could be rejected because: 1) supercomplexes can be isolated from many organisms in an active form; 2) supercomplexes have been proven to stabilize the individual complexes in some cases; 3) supercomplexes can be very stable after chromatographic isolation in some cases....
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics, and play primary roles in the homeostasis of intracellular pH, cellular Na+ content and cell volume. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of such Na+/H+ antiporters. NhaA is the essential Na+/H+ antiporter for pH and Na+ homeostasis (at alkaline pH) in Escherichia coli and many other enterobacteria. NhaA is an electrogenic Na+/H+ antiporter that exchanges 2H+ for 1Na+ (or Li+). NhaA shares with many other prokaryotic and eukaryotic antiporters a very strong dependence on pH. In order to achieve three-dimensional structure of NhaA, the previously described NhaA protein preparation was modified: (i) the wild type bacterial strain (TA16) used for homologous over-expression of NhaA was replaced with a delta nhaA strain (RK20). As a result, the purity and homogeneity of the sample was significantly improved; (ii) the previously two-step purification procedure was shortened to a single step affinity chromatography purification; (iii) a wide-range screening of crystallisation conditions, more than 20,000, was performed; (iv) a Seleno-L-methionine (SeMet) NhaA derivative was produced in order to solve the phases during structure determination. In parallel, attempts of production and crystallisation of co-complexes composed of NhaA and antibody fragments have been made. Four different monoclonal antibodies were available against NhaA. Selected antibody fragments were produced and the stability of the complex analysed. Here, the crystal structure of the pH down-regulated secondary transporter NhaA of Escherichia coli is presented at 3.45 Å resolution. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. A possible mechanism is proposed: the binding of charged substrates causes electric imbalance inducing movements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry. The structure represents a novel fold that provides two major insights: it reveals the structural basis for the mechanism of Na+/H+ exchange and its unique regulation by pH in NhaA and in many other similar antiporters. Furthermore, it is also important for the understanding of the architecture of membrane proteins in general. However, although many aspects of the ion-translocation mechanism and pH regulation are clarified by the NhaA structure, higher resolution structures with Li+ or Na+ bound are required for understanding the ligand binding and the translocation mechanism at the atomic level. The alkaline pH-induced conformation is essential to further understand the pH-control and proton access to the binding site.
Funktionelle und strukturelle Charakterisierung von SLC-Transportern in eukaryotischen Systemen
(2018)
Die evolutionäre Voraussetzung für die Entwicklung komplexer, differenzierter Organismen bildet die Separierung der Zelle in Reaktionsräume, die so genannte Kompartimentierung. Das Prinzip der Kompartimentierung ermöglicht zahlreiche lebensnotwendige, biochemische Prozesse, wie die Konservierung von Energie durch Protonengradienten in der Atmungskette oder parallele, gegenläufige Stoffwechselwege. Zelluläre Kompartimente werden häufig durch Biomembranen gebildet, welche aus einer zweilagigen Lipidschicht bestehen. Lipidmoleküle in einer Zelle sind meistens amphipathisch, das bedeutet, sie bestehen aus einer polaren, hydrophilen Kopfgruppe und einem unpolaren, hydrophopen Ende (Abbildung 1). Die Lipidzusammensetzung in einer Biomembran ist sehr divers und unterscheidet sich in verschiedenen Organismen und Organellen. Phosphoglyceride bilden den Hauptbestandteil der Lipidschicht. Phosphoglyceride besteht aus einem Glycerin Rückgrat, welches an dem C1- und C2-Atom mit zwei Fettsäuren verestert und an dem C3-Atom mit einem Phosphorsäurediester verbunden ist. ...
NADH:ubiquinone oxidoreductase (Complex Ⅰ) is the first and largest enzyme in the respiratory chain. It catalyzes the transfer of two electrons from NADH to ubiquinone via a series of enzyme-bound redox centers - Flavin mononucleotide (FMN) and iron-sulfur (Fe-S) clusters – and couples the exergonic reaction with the endergonic translocation of four protons across the membranes. Bacteria contain the minimal form of complex I, which is composed of 14 conserved core subunits with a molecular mass of around 550 kDa. Complex Ⅰ has an L-shaped structure which can be subdivided into two major parts (arms). The hydrophilic arm protruding into the bacterial cytosol (or mitochondrial matrix) harbors the binding site for the substrate NADH, the two- to one-electron switch FMN and all one-electron transferring Fe-S clusters and therefore considered as the catalytic unit. The membrane arm consists of the membranespanning subunits and conducts the proton pumping process. The Quinone binding site is located at the interface of both arms. ...