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The increasing resistance of almost all pathogenic bacteria to antibiotics (multidrug resistance) causes a severe threat to public health. The mechanisms underlying multidrug resistance include the induced over expression of multidrug transporters which extrude a variety of lipophilic and toxic substrates in an energy dependent fashion through the membrane out of the cell. These proteins are found in all transporter families. The work described in this thesis is dedicated to drug-proton antiporters from the small multidrug resistance (SMR) family. These efflux pumps with just four transmembrane helices per monomer are so far the smallest transporters discovered. Their oligomeric state, topology, three dimensional structure, catalytic cycle and transport mechanism are still rather controversial. Therefore, the aim of this thesis was to directly address these questions for the small multidrug resistance proteins Halobacterium salinarium Hsmr and Escherichia coli (E. coli) EmrE using a number of biophysical methods such as NMR, transport assays, mass spectrometry and analytical ultracentrifugation. Especially the work on Hsmr has been challenging due to the halophilic nature of this protein. In Chapter 1, key questions and the most important biophysical techniques are introduced followed by Material and Methods in Chapter 2. Depending on experimental requirements, cell free or ‘classical’ in vivo expression has been used for this thesis. Cell free expression as an option for the production of small multidrug transporters has been explored in Chapter 3. It has been possible to produce the SMR family members Hsmr, EmrE, TBsmr and YdgF in vitro. The expression of Hsmr was investigated in more detail under different experimental conditions. Hsmr was either refolded from precipitate or maintained in a soluble form during expression in the presence of detergents and liposomes. Furthermore, amino acids for which no auxotrophic strains were available could be labelled successfully. This expression system has been also used for preparing labelled samples of EmrE as described in Chapter 9. In vivo in E. coli expression of Hsmr, as described in Chapter 4, provided large amounts of proteins if fermenter production was used. Uniform labelling and selective unlabelling with stable isotopes (13C, 15N) for NMR spectroscopy was achieved in vivo in a more efficient and cost effective manner than using the cell free approach for this protein. Hsmr could be purified successfully from both in vitro and in vivo expression media. Hsmr is expressed in vivo and in vitro with N-terminal formylation. The Nterminal formylation is unstable and Hsmr in the presence of low salt concentrations was amenable to N-terminal degradation. It was found that Hsmr shows longest stability in Fos-ß-choline® 12 and sodium dodecyl sulphate, but best reconstitution conditions were found, when dodecyl maltoside is used and exchanged with Escherichia coli lipids. A molar protein lipid ratio of 1 to 100, amenable to solid state nuclear magnetic resonance, has been achieved. Sample homogeneity was shown by freeze fracture electron microscopy. The oligomeric state of Hsmr in detergent has been assessed by SDS PAGE, blue native PAGE, size exclusion chromatography, analytical ultracentrifugation and laser induced liquid bead ion desorption mass spectrometry (LILBID) as described in Chapter 5. A concentration and detergent dependent monomer-oligomer equilibrium has been found by all methods. The activity of Hsmr under the sample preparation conditions used here was shown using radioactive and fluorescence binding as well as fluorescence and electrochemical transport assays (Chapter 6). For transport studies, a stable pH gradient was generated by co-reconstitution of Hsmr with bacteriorhodopsin and subsequent sample illumination. Based on the observed long term stability of Hsmr in Fos-ß-choline® 12 and sodium dodecyl sulphate, liquid state NMR experiments were attempted in order to assess the correct folding of Hsmr in detergent micelles (Chapter 7). 1D proton and 2D HSQC spectra of U-15N Hsmr revealed a poor spectral dispersion, low resolution and only a small number of peaks. These are at least partly due to long rotational correlation times of the large protein detergent complex. This problem has been overcome by applying solid-state NMR to Hsmr reconstituted into E. coli lipids (Chapter 8). Uniform 13C labelled samples were prepared and two dimensional proton-driven spin diffusion and double quantum-single quantum correlation spectra were acquired successfully. Unfortunately, the spectral resolution was not yet sufficient for further structural studies. Reasons for the observed linebroadening could be structural heterogeneity or molecular motions which interfere with the NMR timescale. Therefore, the protein mobility has been probed using static 2H solid state NMR on Ala-d3-Hsmr. It could be shown, that parts of Hsmr are remarkably mobile in the membrane and that this mobility can be limited by the addition of the substrate ethidium bromide. Ethidium bromide as well as tetraphenylphosphonium (TPP+) is typical multidrug transporter substrates. The membrane interaction of TPP+ in DMPC membranes has been resolved by 1H MAS NMR. It was found that it penetrates into the interface region of the lipid bilayers and therefore behaves like many other transporter substrates adding to the hypothesis that the membrane could act as a pre-sorting filter. Finally, Chapter 9 is dedicated to the characterisation of the essential and highly conserved residue Glu-14 in EmrE by solid-state NMR. In order to avoid spectral overlap, the single Glu EmrE E25A mutant was chosen instead of the wildtype. The protein has been produced in vitro to take advantage of reduced isotope scrambling in the cell free expression system as verified by analytical NMR spectroscopy. Correct labelling of EmrE was tested by MALDI-TOF and solid-state NMR. The dimeric state of DDM solubilised EmrE has been probed by LILBID. The labelled protein was reconstituted into E. coli lipids to ensure a native membrane environment. Activity was determined by measuring ethidium bromide transport. Freeze fracture EM revealed very homogeneous protein incorporation even after many days of MAS NMR experiments. 2D 13C double quantum filtered experiments were used to obtain chemical shift and lineshape information of Glu-14 in EmrE. Two distinct populations were found with backbone chemical shift differences of 4 - 6 ppm which change upon substrate binding. These findings indicate a structural asymmetry at the assumed dimerisation interface and are discussed in the context of a model for shared substrate/proton binding. These studies represent the first successful use of cell free expression to prepare labelled membrane proteins for solid-state NMR and allow for the first time an NMR insight into the binding pocket of a multidrug efflux pump.
Antibiotic resistance of pathogenic bacteria is a major worldwide problem. Bacteria can resist antibiotics by active efflux due to multidrug efflux pumps. The focus of this study has been the mycobacterial multidrug transporter TBsmr because it belongs to the small multidrug resistance (SMR) family whose members are a paradigm to study multidrug efflux due to their small size. SMR proteins are typically 11-12 kDa in size and have a four-transmembrane helix topology. They bind cationic, lipophilic antibiotics such as ethidium bromide (EtBr) and TPP+, and transport them across the membrane in exchange for protons. To understand the molecular mechanism of multidrug resistance, we have to gain information about the structure and function of these proteins. The research described in this thesis aimed to deduce details about the topology, transport cycle and key residues of TBsmr using biophysical techniques. Solid-state NMR (ssNMR) can provide detailed insight into structural organization and dynamical properties of these systems. However, a major bottleneck is the preparation of mg amounts of isotope labeled protein. In case of proteoliposomes, the problem is compounded by the presence of lipids which have to fit into the small active volume of the ssNMR rotor. In Chapter 3, an enhanced protein preparation is described which yields large amounts of TBsmr reconstituted in a native lipid environment suitable for further functional and structual studies. The achieved high protein-to-lipid ratios made a further characterization by ssNMR feasible. The transport activity and oligomeric state of the reconstituted protein in different types of lipid was studied as shown in Chapter 4. The exact oligomeric state of native SMR proteins is still uncertain but a number of biochemical and biophysical studies in detergent suggest that the minimal functional unit capable of binding substrate is a dimer. However, binding assays are not ideal since a protein may bind substrate without completing the transport cycle which can only be shown for reconstituted protein in transport assays.By combining functional data of a TPP+ transport assay with information about theoligomeric state of reconstituted TBsmr obtained by freeze-fracture electron microscopy, it could be shown that lipids affect the function and the oligomeric state of the protein, and that the TBsmr dimer is the minimal functional unit necessary for transport. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. In Chapter 5, the existence of such a substrate-transporter complex for the TBsmr and its substrate EtBr could be shown. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. The measurements have shown the formation of a pH-dependant, transient substrate-protein complex between binding and release of EtBr. This state was further characterized by determining the Kd, by inhibiting EtBr transport through titration with non-fluorescent substrate and by fluorescence anisotropy measurements. The findings support a model with a single occluded intermediate state in which the substrate is highly immobile. Liquid-state NMR is a useful tool to monitor protein-ligand interactions by chemical shift mapping and thus identify and characterize important residues in the protein which are involved in substrate binding. In agreement with previous studies (Krueger-Koplin et al., 2004), the detergent LPPG was found to be highly suitable for liquid-state NMR studies of the membrane protein TBsmr and 42% of the residues could be assigned, as reported in Chapter 6. However, no specific interactions with EtBr were found. This observation was confirmed by LILBID mass spectrometry which showed that TBsmr was predominantly in the non-functional monomeric state. Functional protein was prepared in proteoliposomes which can be investigated by solidstate NMR (Chapter 7). Besides the essential E13, the aromatic residues W63, Y40, and Y60 have been shown to be directly involved in drug binding and transport. Different isotope labeling strategies were evaluated to improve the quality of the NMR spectra to identify and characterize these key residues. In a single tryptophan mutant of reconstituted TBsmr W30A, the binding of ethidium bromide could be detected by 13C solid-state NMR. The measurements have revealed two populations of the conserved W63 residue with distinct backbone structures in the presence of substrate. There is a controversy about the parallel or anti-parallel arrangement of the protomers in the EmrE dimer (Schuldiner, 2007) but this structural asymmetry is consistent with both a parallel and anti-parallel topology.
The respiratory chain is composed of protein complexes residing in the inner mitochondrial membrane of eukaryotes or in the cytoplasmic membrane of prokaryotes. This cellular energy converter transforms a redox potential stored in low potential substrates into an electrochemical potential across the respective membrane. Typical respiratory chains contain the complexes I, II, III and IV named according to their sequence in the respiratory chain reaction. Electrons of low potential substrates enter at complex I or II and are passed via complex III to complex IV where they are transferred to oxygen. The transport of electrons between the complexes is mediated by small electron shuttles like quinol or cytochrome c. Two different models describe their exchange either by (1) random collision of freely diffusible electron shuttles and membrane protein complexes or (2) arrangement of the complexes in supercomplexes enabling direct channeling of electron shuttles. In the Gram positive bacterium Corynebacterium glutamicum, the complex III to complex IV electron shuttle cytochrome c is not diffusible but a covalently bound part of the diheme cytochrome subunit QcrC of complex III. Therefore, the complexes III and IV have to form a supercomplex for electron transduction. The aim of this thesis was to purify and characterise this obligatory supercomplex III/IV of C. glutamicum. To gain sufficient biomass of C. glutamicum as starting material for purification, a phosphate buffered minimal medium was developed that enabled yield of total 120 g wet cell mass (38 g dry mass) in 12 L (6×2 L) shaking cultures. The determined conversion factor of glucose into biomass was 0.46 g/g indicating an intact respiratory chain. The yield was increased by bioreactor cultivation to ~690 g wet cell mass (~220 g dry mass) in ~10 L culture volume. A previously described homologous expression system was applied that produces the complex IV subunit CtaD with a fused Strep-tag II to facilitate purification. Affinity purifications using the Strep-tag II affinity to Strep-Tactin resin yielded a mixture of complexes and supercomplexes. Two supercomplex III/IV versions named supercomplex A and B and free complex IV were identified in this mixture by size exclusion chromatography, redox difference spectroscopy and two dimensional polyacrylamide gel electrophoresis including blue native polyacrylamide electrophoresis. The here presented downscaled blue native polyacrylamide electrophoresis method with analysis times of ~1 h enabled efficient screening of factors influencing the stability of supercomplex III/IV. The screening resulted that the integrity of supercomplex III/IV is preserved by using neutral detergents at minimal detergent to protein ratios for solubilisation and low detergent concentrations for purification and storage slightly above the required critical micellar concentration. Furthermore, pH <=7.5 is required for stability of supercomplex III/IV. Large biomass yields enabled upscaling of supercomplex III/IV affinity purification. Application of the identified stability conditions resulted in affinity purified samples free of supercomplex B. The major component supercomplex A was efficiently separated from residual free complex IV by preparative size exclusion chromatography. Concentration of purified supercomplex A by ultracentrifugation resulted in integrity of the supercomplex for several days at 4 °C. Purified supercomplex A contains ten different previously described subunits. The heme content of supercomplex A relative to the protein mass is heme A: 6.0 μmol/g, heme B: 6.5 μmol/g, and heme C: 5.8 μmol/g determined by redox difference spectroscopy and biochemical protein quantification. This indicates an equimolar ratio of complex III and complex IV in supercomplex A. Supercomplex A has quinol oxidase activity that is inhibited by stigmatellin or sodium azide. The turnover number of transferred electrons per complex III monomer is 148 s−1 at 25° C. The homogeneity and stability of the prepared supercomplex A enabled the growth of threedimensional crystals of up to 0.1 mm in length. Their composition of supercomplex A was verified by redox difference spectroscopy of intact crystals and blue native polyacrylamide electrophoresis of dissolved crystals. The crystals diffracted X-rays corresponding to a resolution of ~10 Å. Electron microscopy of negative stained samples revealed the uniform shape of purified supercomplex A particles with dimensions of 22 × 9 nm in the view plane. Combined heme quantification, size determination, determined activity, symmetry considerations, and particle shape indicate that supercomplex A has a central dimer of complex III and two monomers of complex IV on opposite sides. This conformation is functionally reasonable because it provides each complex III monomer with one complex IV monomer as electron acceptor. Therefore, the stoichiometry of supercomplex A is most likely III2IV2. The sensitivity of supercomplex A to detergents indicated a role of phospholipids in its stability. Therefore, a method for phospholipid identification and quantification was developed that is suitable for detergent solubilised crude and purified membrane protein samples. The analysis combines separation of phospholipid classes according to their head group by normal phase high performance liquid chromatography with evaporative light scattering detection. Calibration with external standard allows quantification of phospholipid amount in the range of 0.25-12 μg. The method is verified by analysing the phospholipid content of the well characterised complex III of Saccharomyces cerevisiae. The reduction of its phospholipid content during its purification steps is monitored. The complex III sample purified to crystallisation quality contains the phospholipid content that was also observed in previously reported structures determined by X-ray crystallography. Purified stable supercomplex A from C. glutamicum revealed a large content of bound phospholipids. The main differences between intact supercomplex A and a mixture of potentially disintegrated smaller complexes is that intact supercomplex A has a doubled phosphatidic acid content and an increased phosphatidyl glycerol content. The importance of the small anionic phosphatidic acid for mediation of contacts between complexes in a supercomplex is discussed. The total phospholipid content of stable supercomplex A is sufficient for a complete belt surrounding the supercomplex in the membrane plane. This indicates that also all essential internal phospholipid binding positions are occupied and potentially stabilise supercomplex A.
In der vorliegenden Arbeit ist es gelungen, OTS-, MPTMS-, und MPTMS/OTS gemischte SAMs aus der Lösung auf SC-1 chemisch oxidierten Siliziumwafern („SiO2“) zu präparieren. Die Adsorption der OTS oder MPTMS SAMs auf SiO2 wird von zwei konkurrierenden Reaktionen bestimmt, d.h. „Selbstaggregation” in der Ausgangslösung und “Oberflächendehydration” des SiO2 -Substrates. Die beiden Alkylsiloxan-SAMs weisen unterschiedliches Bildungsverhalten auf. Die Reifungsdauer der Ausgangslösung vor der Adsorption wirkt sich signifikant auf die Bildung der OTS SAMs aus, demgegenüber ist bei MPTMS SAMs kein Einfluß zu beobachten. Für OTS SAMs sind große Dendriten oftmals von kleinen Rundinseln umgeben, dagegen für MPTMS SAMs treten prinzipiell nur sporadisch verteilte kleine Rundinseln auf. Die Abwesenheit des Chlor-Signals in XPS-Spektren bestätigt, dass innerhalb der Adsorption die Si-Cl Bindungen der OTS-Moleküle zum größten Teil hydrolysiert werden. Doch für MPTMS SAMs ist in C 1s-Spektren ein Peak bei 286.4 eV, der der unhydrolysierten Si-OCH3 Bindung entspricht, zu beobachten. Die Hydrolysefähigkeit der Si-Cl Bindung des OTS ist erwartungsgemäß stärker als jene der Si-OCH3 Bindung des MPTMS. Diese Tendenz samt dem Unterschied in der Alkylkettenlänge wirkt sich beträchtlich auf die Bildung und die Morphologie der adsorbierten Inseln aus. Bei gleicher Konzentration (5 mM) und Reifungsdauer der Ausgangslösung bilden sich OTS SAMs viel schneller als MPTMS SAMs bei Raumtemperatur. Sie hat auch eine größere Oberflächenbedeckung wegen der seitlichen Vernetzung zur Folge. Diese Beobachtung zeigt eine prognostizierbare kinetische Schwierigkeit zur Präparation der OTS/MPTMS gemischten SAMs durch Koadsorption. Grund hierfür ist, dass die Adsorption voraussichtlich von OTS-Molekülen dominiert wird. Darüber hinaus ist Aggregation zwischen hydrolysierten OTS- und MPTMS-Molekülen nicht ausgeschlossen. Neben der Koadsorption steht in der zweistufigen Adsorption ein weiteres herkömmliches Verfahren zur Verfügung. Das Endprodukt kann nach der Reaktionsreihe der Silane mit „OTS+MPTMS“ oder „MPTMS+OTS“ gemischte SAMs bezeichnet werden. Unter Berücksichtigung der individuellen Oberflächenbedeckung und Morphologie wurde eine Rezeptur aufgestellt, in der die Adsorption jeweils höchstens 30s (für OTS) und 20 min (für MPTMS) dauert. Angesichts der vielfältigen Inselstruktur, d.h. Monoschicht, polymerisierte Bälle, und sogar Multischicht, ist eine Phasenunterscheidung nach der Dicke, z.B. mittels AFM, nicht zu erwarten. Die Existenz der lateralen unvernetzten Si-OH Gruppen der adsorbierten OTS-Inseln könnte die Präparation der homogenen OTS+MPTMS gemischten SAMs erschweren. In diesem Fall ist es fraglich, ob die hydrolysierten MPTMS-Moleküle vollständig wie geplant mit oberflächnahen OH-Gruppen von SiO2 reagieren. Mit einer umgekehrten Reaktionsreihe löst sich das Problem von selbst, da die adsorbierten MPTMS-Inseln hauptsächlich unhydrolysierte seitliche Si-OCH3 Gruppen besitzen. Die morphologische Erkennbarkeit unterstützt die Machbarkeit der Präparation der MPTMS+OTS gemischten SAMs. Die unterschiedlichen Messmodi des AFM, mit denen die Morphologie der OTS SAMs aufgenommen wurde, ergaben deutliche Unterschiede in ihrem Erscheinungsbild. Im Vergleich zum Tappingmodus sind die Grenzen der OTS-Inseln auf Kontaktmodus-Bildern nur undeutlich erkennbar. Die großen Inseln erscheinen nicht so dendritisch. Die Ursache dieser Phänomene könnte am Wassermeniskus zwischen Spitze und Probe liegen, da die Messung nicht in Flüssigkeit, sondern an Luft durchgeführt wurde. Auf LFM-Bildern sind die adsorbierten OTS-Inseln heller als unbedecktes SiO2, während die MPTMS-Inseln dunkler als SiO2 aussehen. Eine ähnliche Auswirkung der Messmodi auf die Morphologie der MPTMS SAMs wurde nicht beobachtet. Durch die Adsorption von 1-Decanthiol lässt sich die Si3N4-AFM-Spitze modifizieren. Eine solche CH3-terminierte Spitze ist hydrophob und verursacht einen gegenteiligen Helligkeitskontrast auf LFM-Bildern der adsorbierten OTS-Inseln.
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.