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In this review, we discuss the mechanisms of generation of membrane-bound protons using different energy sources in model and natural systems. Analysis of these mechanisms revealed that all three types of reactions include the same principal stage, which is dissociation of electrically neutral Brønsted acids at the interface during transition from the hydrophobic phase to water with a low dielectric constant. Special attention is paid to the fact that in one of the analyzed model systems, membrane-bound protons provide energy for the reaction of ATP synthesis. Similar mechanism for the generation of membrane-bound protons has been found in natural membranes involved in oxidative phosphorylation, in particular, on the membranes of mitoplasts and mitochondria. The energy of oxidative reactions required for ATP synthesis, is stored at the intermediate stage not only in the form of transmembrane electrochemical potential of protons, but also and perhaps mostly, as protons attached to the inner mitochondrial membrane. The process of energy storage in mitochondria is linked to the transfer of protons that simultaneously perform two functions. Protons on the membrane surface carry free energy and, at the same time, act as substrates facilitating the movement of F1F0-ATP-synthase biological machine. [ABSTRACT FROM AUTHOR]

A depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative 'superbugs' has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of "superbugs". An increasing prevalence of Gram-negative bacteria increases the interest in nanotherapies to treat antibiotic resistance. Here, the authors examine the antimicrobial activity of polymyxin-loaded cubosomes and explore a polytherapy treatment of pathogens with cubosomes in combination with polymyxin. [ABSTRACT FROM AUTHOR]

At the joint between the membrane and hydrophilic arms of the enzyme, the structure of the respiratory complex I reveals a tunnel-like Q-chamber for ubiquinone binding and reduction. The narrow entrance of the quinone chamber located in ND1 subunit forms a bottleneck (eye of a needle) which in all resolved structures was shown to be too small for a bulky quinone to pass through, and it was suggested that a conformational change is required to open the channel. The closed bottleneck appears to be a well-established feature of all structures reported so-far, both for the so-called open and closed states of the enzyme, with no indication of a stable open state of the bottleneck. We propose a squeeze-in mechanism of the bottleneck passage, where dynamic thermal conformational fluctuations allow quinone to get in and out. Here, using molecular dynamics simulations of the bacterial enzyme, we have identified collective conformational changes that open the quinone chamber bottleneck. The model predicts a significant reduction—due to a need for a rare opening of the bottleneck—of the effective bi-molecular rate constant, in line with the available kinetic data. We discuss possible reasons for such a tight control of the quinone passage into the binding chamber and mechanistic consequences for the quinone two-electron reduction. [ABSTRACT FROM AUTHOR]

This study investigated the influence of two polyphenols on the structure and lubrication of the salivary pellicle, aiming to extend the understanding of astringency mechanisms. The salivary pellicle was prepared by the adsorption of human whole saliva on the enamel substrate. Low-astringency catechin and high-astringency tannic acid were used as astringents. The changes induced by the two polyphenols in the structure and lubrication of the salivary pellicle were examined using quartz crystal microbalance with dissipation (QCM-D) and nano-indentation/scratch technique. The salivary pellicle suffers from changes in structure and physical properties owing to protein dehydration and protein-polyphenol complexation when encountering polyphenolic molecules, causing increases in the roughness and contact angle but a decrease in the load-bearing capacity. Therefore, the lubrication performance of the salivary pellicle is impaired, leading to an increase and fluctuation of the friction coefficient. The intensity of astringency has a strong positive correlation with the water contact angle, surface roughness, and friction coefficient of the salivary pellicle. In summary, astringency is a tactile perception driven by the roughness and wettability of the salivary pellicle rather than oral lubrication, and increased intraoral friction is an inevitable consequence of astringency. The findings of this study will help promote and assist the objective evaluation of astringency. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase 6B1 (COX6B1) is one of the less characterized subunits of the mitochondrial electron transport chain complex IV (CIV). Here, we studied the pathobiochemical and respiratory functions of Cox12 (yeast ortholog of COX6B1) using Saccharomyces cerevisiae BY4741 (cox12Δ) cells deficient by the Cox12 protein. The cells exhibited severe growth deficiency in the respiratory glycerol-ethanol medium, which could be reverted by complementation with the yeast COX12 or human COX6B1 genes. Cox12 with arginine 17 residue substituted by histidine (R17H) or cysteine (R17C) (mutations analogous to those observed in human patients) failed to complement the loss of Cox12 function. When cox12Δ cells were grown in rich respiratory/fermentative galactose medium, no changes in the expression of individual respiratory chain subunits were observed. Blue native PAGE/Western blotting analysis using antibodies against Rip1 and Cox1, which are specific components of complexes III (CIII) and IV (CIV), respectively, revealed no noticeable decrease in the native CIII2CIV2 and CIII2CIV1 supercomplexes (SCs). However, the association of the respiratory SC factor 2 (Rcf2) and Cox2 subunit within the SCs of cox12Δ cells was reduced, while the specific activity of CIV was downregulated by 90%. Both basal respiration and succinate-ADP stimulated state 3 respiration, as well as the mitochondrial membrane potential, were decreased in cox12Δ cells. Furthermore, cox12Δ cells and cells synthesizing Cox12 mutants R17H and R17C showed higher sensitivity to the H2O2-induced oxidative stress compared to the wild-type (WT) cells. In silico structural modeling of the WT yeast SCs revealed that Cox12 forms a network of interactions with Rcf2 and Cox2. Together, our results establish that Cox12 is essential for the CIV activity. [ABSTRACT FROM AUTHOR]

Impedance-based protein detection sensors for point-of-care diagnostics require quantitative specificity, as well as rapid or real-time operation. Furthermore, microfabrication of these sensors can lead to the formation of factors suitable for in vivo operation. Herein, we present microfabricated needle-shaped microwell impedance sensors for rapid-sample-to-answer, label-free detection of cytokines, and other biomarkers. The microneedle form factor allows sensors to be utilized in transcutaneous or transvascular sensing applications. In vitro, experimental characterization confirmed sensor specificity and sensitivity to multiple proteins of interest. Mechanical characterization demonstrated sufficient microneedle robustness for transcutaneous insertion, as well as preserved sensor function postinsertion. We further utilized these sensors to carry out real-time in vivo quantification of human interleukin 8 (hIL8) concentration levels in the blood of transgenic mice that endogenously express hIL8. To assess sensor functionality, hIL8 concentration levels in serum samples from the same mice were quantified by ELISA. Excellent agreement between real-time in vivo sensor readings in blood and subsequent ELISA serum assays was observed over multiple transgenic mice expressing hIL8 concentrations from 62 pg/mL to 539 ng/mL. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion. Thus, the enzyme's catalytic cycle may have to be turned by 180 degrees. Cytochrome c oxidase is a fundamental enzyme of life and its mechanism is not fully understood yet. Here, the authors present four cryo-EM structures of different intermediate states, which suggest an alternative cytochrome c oxidase reaction cycle. [ABSTRACT FROM AUTHOR]

The effect of exogenous cytochrome c (cyt c) on kinetics of photoelectric responses (Δψ) of two types of photosystem II (PSII) core complexes (intact – PSII with active water-oxidizing complex and Mn-depleted complex) reconstituted into liposomes has been investigated by direct electrometric technique. PSII complexes were localized in the proteoliposome membranes with their donor side outward. An additional electrogenic phase was observed in the kinetics of Δψ generation in response to a laser flash besides the main fast (<0.3 µs) electrogenic component due to electron transfer from the redox-active tyrosine YZ to the primary quinone acceptor QA in the presence of oxidized cyt c (cyt c3+) entrapped in the internal space of proteoliposomes with intact PSII complexes. This component with characteristic time τ ≈ 40 µs and relative amplitude of ~10% of the total Δψ was attributed to the vectorial electron transfer from QA− to cyt c3+ serving as an external acceptor. An additional electrogenic component with τ ~ 70 µs and a relative amplitude of ~20% of the total Δψ also appeared in the kinetics of Δψ formation, when cyt c2+ was added to the suspension of proteoliposomes containing Mn-depleted PSII core complexes. This component was attributed to the electrogenic transfer of an electron from cyt c2+ to photooxidized tyrosine YZ. These data imply that cyt c3+ serves as a very effective exogenous electron acceptor for QA− in the case of intact PSII core complexes, and cyt c2+ is an extremely efficient artificial electron donor for YZ in the Mn-depleted PSII. The obtained data on the roles of cyt c2+ and cyt c3+ as an electron donor and acceptor for PSII, respectively, can be used to develop hybrid photoelectrochemical solar energy-converting systems based on photosynthetic pigment–protein complexes. [ABSTRACT FROM AUTHOR]

The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes1, whose precise role is debated. Supercomplexes CIII2CIV1-2 (refs. 2,3), CICIII2 (ref. 4) and CICIII2CIV (respirasome)5–10 exist in mammals, but in contrast to CICIII2 and the respirasome, to date the only known eukaryotic structures of CIII2CIV1-2 come from Saccharomyces cerevisiae11,12 and plants13, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII2CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII2CIV from the CIII2 precursor to the final CIII2CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. 14) deep into CIII2, while its C terminus is integrated into CIV. Our structures (which include CICIII2 and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII2CIV and has no role in the assembly of the respirasome. We show that CIII2 is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII2, explaining the presence of one copy of CIV in CIII2CIV in mammals. Finally, we show that CIII2 and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII2CIV in fine tuning the efficiency of electron transfer in the electron transport chain.SCAF1 is solely required for supercomplex CIII2CIV assembly and is not involved in the formation of the respirasome (supercomplex CICIII2CIV) [ABSTRACT FROM AUTHOR]

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells. A variety of artificial cells springs from the functionalization of liposomes with proteins but these models suffer from low durability without repair and replenishment mechanisms. Here, the authors show that synthetic amphiphile membranes undergo SNARE-mediated fusion, and determine bending rigidity and pore edge tension as key parameters for fusion. [ABSTRACT FROM AUTHOR]

Systematic collections of single-gene deletions have been invaluable in uncovering determinants of lifespan in yeast. Overexpression of a single gene does not have such a clear outcome as cancellation of its function but it can lead to a variety of imbalances, deregulations and compensations, and some of them could be important for longevity. We report an experiment in which a genome-wide collection of strains overexpressing a single gene was assayed for chronological lifespan (CLS). Only one group of proteins, those locating to the inner membrane and matrix of mitochondria, tended to extend CLS when abundantly overproduced. We selected two such strains—one overexpressing Qcr7 of the respiratory complex III, the other overexpressing Mrps28 of the small mitoribosomal subunit—and analyzed their transcriptomes. The uncovered shifts in RNA abundance in the two strains were nearly identical and highly suggestive. They implied a distortion in the co-translational assembly of respiratory complexes followed by retrograde signaling to the nucleus. The consequent reprogramming of the entire cellular metabolism towards the resistance to stress resulted in an enhanced ability to persist in a non-proliferating state. Our results show that surveillance of the inner mitochondrial membrane integrity is of outstanding importance for the cell. They also demonstrate that overexpression of single genes could be used effectively to elucidate the mitochondrion-nucleus crosstalk. [ABSTRACT FROM AUTHOR]

Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor. Zabelskii et al. present a structural and functional analysis of the lightdriven proton pump LR (Mac) from the fungus Leptosphaeria maculans. Their findings indicate that the archaeal ancestry of eukaryotic type 1 rhodopsins, and that the archaeal host of the proto-mitochondrial endosymbiont was capable of light-driven proton pumping. [ABSTRACT FROM AUTHOR]

NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients' tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253AMT-ND5 mutation completely disturbed the assembly of complex I, while the mutation D199GMT-ND1 led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199MT-ND1 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199MT-ND1 for energy conservation in complex I is discussed. [ABSTRACT FROM AUTHOR]

The characteristics of cultured cell attachment onto poly-l-lysine (PLL), collagen, and the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) were studied using a quartz crystal microbalance (QCM). A QCM with microscope cameras enclosed in a Peltier chamber was developed to enable QCM measurements and microphotographic imaging to be conducted in a temperature-controlled CO2 incubator. Human hepatoma cell line HepG2 cells were cultured on the quartz crystals coated with PLL, collagen, and PNIPAM. Response curves of the resonant frequency of the quartz crystals during the cell attachment process were analyzed on the basis of the parameters of modeling curves fit to the experimentally obtained curves. Analysis of the fitting curves showed that the time constants of the first-lag response were 11 h for PLL, 16 h for collagen, and 38 h for PNIPAM and that the frequency change for the PNIPAM films was six times smaller than those for the PLL and collagen films. These findings were supported by photographic images showing wider cell spread on PLL and collagen than on PNIPAM. The response of cells on PNIPAM was measured during a thermal cycle from 37 to 20 °C to 37 °C. In the resonance frequency–resonance resistance (F–R) diagram, the slopes of ΔR/ΔF corresponding to the cell attachment process and those corresponding to the thermal cycling process differed; the positions in the F–R diagram also shifted to higher resonant frequencies after the thermal cycle. These results suggested that the mass effect decreased as a result of the weakening of the cell attachment strength by the thermal cycle because the molecular brushes of PNIPAM were disarranged. [ABSTRACT FROM AUTHOR]

Electrons from cytochrome c, the substrate of cytochrome oxidase, a redox-linked proton pump, are accepted by CuA in subunit II. From there they are transferred to the proton pumping machinery in subunit I, cytochrome a and cytochrome a3–CuB. The reduction of the latter site, which is the dioxygen reducing unit, is coupled to proton uptake. Dioxygen reduction involves a peroxide and a ferryl ion intermediate, and it is the transition between these and back to the resting oxidized enzyme that are coupled to proton pumping. The X-ray structures suggest electron–transfer pathways that can account for the observed rates provided that the reorganization energies are small. They also reveal two proton-transfer pathways, and mutagenesis experiments have shown that one is used for proton uptake during the initial reduction of cytochrome a3–CuB, whereas the other mediates transfer of the pumped protons. [ABSTRACT FROM AUTHOR]

During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a3, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O2 reduction. [ABSTRACT FROM AUTHOR]

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Copyright of Der Unfallchirurg is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria. Respiratory chains generate the proton motive force used for ATP synthesis. Cryo-EM structures of functional respiratory CIII2CIV supercomplex and native CIII2 from Rhodobacter capsulatus provide insight into CIII2CIV assembly and respiratory electron transport pathways in Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Let m , n ∈ N and p ∈ (0 , ∞) . For a finite dimensional quasi-normed space X = (R m , ‖ · ‖ X) , let B p n (X) = (x 1 , ... , x n) ∈ ( R m ) n : ∑ i = 1 n ‖ x i ‖ X p ⩽ 1. We show that for every p ∈ (0 , 2) and X which admits an isometric embedding into L p , the function S n - 1 ∋ θ = (θ 1 , ... , θ n) ⟼ B p n (X) ∩ (x 1 , ... , x n) ∈ ( R m ) n : ∑ i = 1 n θ i x i = 0 is a Schur convex function of (θ 1 2 , ... , θ n 2) , where | · | denotes Lebesgue measure. In particular, it is minimized when θ = (1 n , ... , 1 n ) and maximized when θ = (1 , 0 , ... , 0) . This is a consequence of a more general statement about Laplace transforms of norms of suitable Gaussian random vectors which also implies dual estimates for the mean width of projections of the polar body (B p n (X)) ∘ if the unit ball B X of X is in Lewis' position. Finally, we prove a lower bound for the volume of projections of B ∞ n (X) , where X = (R m , ‖ · ‖ X) is an arbitrary quasi-normed space. [ABSTRACT FROM AUTHOR]

Cardiolipin (CL), a crucial component in inner mitochondrial membranes, interacts with cytochrome c (cyt c) to form a peroxidase complex for the catalysis of CL oxidation. Such interaction is pivotal to the mitochondrial regulation of apoptosis and is affected by the redox state of cyt c. In the present study, the redox-dependent interaction of cyt c with CL was investigated through amide hydrogen/deuterium exchange coupled with mass spectrometry (HDXMS) and quartz crystal microbalance with dissipation monitoring (QCM-D). Ferrous cyt c exhibited a more compact conformation compared with its ferric form, which was supported by the lower number of deuterons accumulated and the greater amplitude reduction on dissipation. Upon association with CL, ferrous cyt c resulted in a moderate increase in deuteration, whereas the ferric form caused a drastic increase of deuteration, which indicated that CL-bound ferric cyt c formed an extended conformation. These results were consistent with those of the frequency (f) − dissipation (D) experiments, which revealed that ferric cyt c yielded greater values of |ΔD/Δf| within the first minute. Further fragmentation analysis based on HDXMS indicated that the effect of CL binding was considerably different on ferric and ferrous cyt c in the C-helix and the Loop 9–24. In ferric cyt c, CL binding affected Met80 and destabilized His18 interaction with heme, which was not observed with ferrous cyt c. An interaction model was proposed to explain the aforementioned results. [ABSTRACT FROM AUTHOR]

Mitochondria are recognized as the main source of ATP to meet the energy demands of the cell. ATP production occurs by oxidative phosphorylation when electrons are transported through the electron transport chain (ETC) complexes and develop the proton motive force across the inner mitochondrial membrane that is used for ATP synthesis. Studies since the 1960s have been concentrated on the two models of structural organization of ETC complexes known as "solid-state" and "fluid-state" models. However, advanced new techniques such as blue-native gel electrophoresis, mass spectroscopy, and cryogenic electron microscopy for analysis of macromolecular protein complexes provided new data in favor of the solid-state model. According to this model, individual ETC complexes are assembled into macromolecular structures known as respiratory supercomplexes (SCs). A large number of studies over the last 20 years proposed the potential role of SCs to facilitate substrate channeling, maintain the integrity of individual ETC complexes, reduce electron leakage and production of reactive oxygen species, and prevent excessive and random aggregation of proteins in the inner mitochondrial membrane. However, many other studies have challenged the proposed functional role of SCs. Recently, a third model known as the "plasticity" model was proposed that partly reconciles both "solid-state" and "fluid-state" models. According to the "plasticity" model, respiratory SCs can co-exist with the individual ETC complexes. To date, the physiological role of SCs remains unknown, although several studies using tissue samples of patients or animal/cell models of human diseases revealed an associative link between functional changes and the disintegration of SC assembly. This review summarizes and discusses previous studies on the mechanisms and regulation of SC assembly under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

The effect of Zn2+ on the P-side of proteoliposomes containing membrane-incorporated Rhodobacter sphaeroides cytochrome c oxidase was investigated by the time-resolved electrometrics following a single electron injection into the enzyme prepared in the F state. The wild-type enzyme was examined along with the two mutants, N139D and D132N. All obtained data indicate that the primary effect of Zn2+ added from the P-side of the membrane is slowing of the pumped proton release from the proton loading site (PLS) to the bulk aqueous phase on the P-side of the membrane. The results strongly suggest the presence of two pathways by which the pumped proton can exit the protein from the PLS and of two separate binding sites for Zn2+. A model is presented to explain the influence of Zn2+ on the kinetics of membrane-potential generation by the wild-type COX, as well as by the N139D and D132N mutants. [ABSTRACT FROM AUTHOR]

Several ferryl states of the catalytic heme a3-CuB center of the respiratory cytochrome c oxidases (CcOs) are observed during the reduction of O2 to H2O. One of the P-type ferryl forms, PM, is produced by the reaction of the two-electron reduced CcO with O2. In this state, the heme a3 iron is in the ferryl state and a free radical should be also present at the catalytic center. However, the energetics of the PM formation has not been experimentally established yet. Here, the generation of PM by the reaction of oxidized bovine CcO (O) with one molecule of H2O2 was investigated by the isothermal titration calorimetry and UV-Vis absorption spectroscopy. Two kinetic phases, corresponding to the formation of PM and its endogenous conversion back to O, were resolved by both methods. The ΔH of the entire process (–66 kcal/mol H2O2) was larger than the heat (–50.8 kcal/mol O2) liberated during O2 reduction by ferrocytochrome c (pH 8, 25°C). Interestingly, ΔH of the first phase (–32 kcal/mol ferryl state) far exceeds the enthalpy of the PM production. The data indicate that during the first phase, the radical in PM is quenched and spectrally similar second P-type ferryl form (PR) is produced. Additionally, it was shown that the entropy contribution to the Gibbs energy change (ΔG = –46 kcal/mol O2) during the catalytic reduction of O2 by ferrocytochrome c is negligible (–0.7 cal·mol–1·K–1). [ABSTRACT FROM AUTHOR]

2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the most commonly used herbicides worldwide. While the effects of 2,4-D in target organisms are well known, its consequences in nontarget organisms are not fully explained. Therefore, the purpose of this study was to investigate the effects of the herbicide on mitochondrial energy metabolism, oxidative status, and exploratory behavior in adult zebrafish. Animal exposure to 2,4-D increased cytochrome c oxidase and catalase activities and reduced SOD/CAT ratio, moreover, increased the total distance traveled and the number of crossings. Finally, animals exposed to 2,4-D spent more time in the upper zone of the tank and traveled a long distance in the upper zone. Overall, our results indicate the 2,4-D can provoke disabling effects in nontarget organisms. The obtained data showed that exposure to 2,4-D at environmentally relevant concentrations alters mitochondrial metabolism and antioxidant status and disturbs the zebrafish innate behavior. [ABSTRACT FROM AUTHOR]

The light-induced functioning of photosynthetic pigment-protein complex of photosystem II (PSII) is linked to the vectorial translocation of charges across the membrane, which results in the formation of voltage. Direct measurement of the light-induced voltage (∆V) generated by spinach oxygen-evolving PSII core complexes adsorbed onto a Millipore membrane filter (MF) on an indium tin oxide (ITO) electrode under continuous illumination has been performed. PSII was shown to participate in electron transfer from water to the ITO electrode, resulting in ∆V generation. No photovoltage was detected in PSII deprived of the water-oxidizing complex. The maximal and stable photoelectric signal was observed in the presence of disaccharide trehalose and 2,6-dichloro-1,4-benzoquinone, acting as a redox mediator between the primary quinone acceptor QA of PSII and electrode surface. Long time preservation of the steady-state photoactivity at room temperature in a simple in design ITO|PSII-MF|ITO system may be related to the retention of water molecules attached to the PSII surface in the presence of trehalose. [ABSTRACT FROM AUTHOR]

In this study, cellulose nanofibers (CNFs) from softwood and hardwood kraft pulps were prepared with different hemicellulose contents, and the effects of the hemicellulose compositions and contents on the interaction between CNFs were studied. In addition, the effects of CNF interaction on the CNF dispersion stability and viscosity characteristics in the aqueous state were investigated. Using quartz crystal microbalance with dissipation, the results of the interaction analysis revealed that hemicellulose moderated the strength of the interaction between CNFs, and the intensity of the interaction changed according to the hemicellulose compositions. The interaction between galactoglucomannans was stronger than that between glucuronoxylans, and CNFs prepared from softwood pulp containing mainly galactoglucomannan showed stronger interaction between CNFs than those prepared from hardwood pulp containing mainly glucuronoxylan. The presence of hemicelluloses on the surface of CNFs improved the dispersion stability and increased the viscosity. However, the intensity of the interaction between CNFs, which is dependent on the type of hemicellulose, did not affect these properties. The properties of CNFs in the aqueous state are determined by not only the interaction between CNFs but also several factors such as the morphology of CNFs and physical properties of hemicelluloses. [ABSTRACT FROM AUTHOR]

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions. [ABSTRACT FROM AUTHOR]

In cytochrome c oxidase electron transfer from cytochrome c to O2 is linked to transmembrane proton pumping, which contributes to maintaining a proton electrochemical gradient across the membrane. The mechanism by which cytochrome c oxidase couples the exergonic electron transfer to the endergonic proton translocation is not known, but it presumably involves local structural changes that control the alternating proton access to the two sides of the membrane. Such redox-induced structural changes have been observed in X-ray crystallographic studies at residues 423-425 (in the R. sphaeroides oxidase), located near heme a. The aim of the present study is to investigate the functional effects of these structural changes on reaction steps associated with proton pumping. Residue Ser425 was modified using site-directed mutagenesis and time-resolved spectroscopy was used to investigate coupled electron-proton transfer upon reaction of the oxidase with O2. The data indicate that the structural change at position 425 propagates to the D proton pathway, which suggests a link between redox changes at heme a and modulation of intramolecular proton-transfer rates. [ABSTRACT FROM AUTHOR]

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The widely used mood stabilizer valproate (VPA) causes perturbation of energy metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. To gain insight into these mechanisms, we determined the effects of VPA on energy metabolism in yeast. VPA treatment increased levels of glycolytic intermediates, increased expression of glycolysis genes, and increased ethanol production. Increased glycolysis was likely a response to perturbation of mitochondrial function, as reflected in decreased membrane potential and oxygen consumption. Interestingly, yeast, mouse liver, and isolated bovine cytochrome c oxidase were directly inhibited by the drug, while activities of other oxidative phosphorylation complexes (III and V) were not affected. These findings have implications for mechanisms of therapeutic action and toxicity. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) is a multi-factorial bone disease influenced by genetic factors, age, and lifestyles. The aim of this study is to evaluate the genetic correlations between OP and multiple lifestyle-related factors, and explore the genes underlying the detected genetic correlations. Linkage disequilibrium score regression (LDSC) analysis was applied to evaluate the genetic correlations of total body bone mineral density (TB-BMD) of different ages (including 15-30 years, 30-45 years, 45-60 years, and over 60 years) with four common lifestyle/environment-related factors (including serum 25-hydroxyvitamin D, cigarette smoking, alcohol dependence, and caffeine metabolites). Transcriptome-wide association studies (TWAS) of TB-BMD (30-45 years) and smoking were conducted in peripheral blood (PB), whole blood (WB), and adipose tissues. The identified candidate genes were also subjected to gene set enrichment analysis (GSEA). Genetic correlation was only observed between TB-BMD (30-45 years) and cigarette smoking status (P = 0.01, LD score = 0.11 ± 0.04). No significant genetic correlation was detected for other lifestyle/environmental factors, including serum 25-hydroxyvitamin D, alcohol dependence, and caffeine metabolites for TB-BMD within all of the four age groups. TWAS identified 85 genes in PB and 163 genes in WB for TB-BMD, as well as 123 genes in PB and 257 genes in WB for smoking. Multiple common candidate genes shared by both TB-BMD and smoking were detected, such as MAP1LC3B (PTB-BMD-PB = 1.00 × 10-3, Psmoking-PB = 9.62 × 10-3, PTB-BMD-WB = 2.99 × 10-2) and SLC23A3 (PTB-BMD-WB = 1.48 × 10-2, Psmoking-WB = 8.76 × 10-3). GSEA detected one GO terms for TB-BMD (cytosol) in WB, one GO term for smoking (mitochondrion) in PB, and one pathway (oocyte meiosis) for smoking in WB. [ABSTRACT FROM AUTHOR]

Surface adsorption of amphiphilic molecules is a vital mechanism of boundary lubrication on stainless steel surfaces. The self-assembly of four fatty acid-based organic friction modifiers in two alkane solvents and their adsorption onto stainless steel surfaces was investigated using Dynamic Light Scattering and Quartz Crystal Balance with Dissipation, respectively. These properties were related to the friction force between a sharp tip and the steel surface measured using Lateral Force Microscopy. The molecular structures of the organic friction modifiers were chosen in order to study the effects of unsaturation and number of alkyl chains as well as the composition of the polar head groups on their assembly in solution, adsorption, and nanotribological behavior. Sorbitan monooleate and dioleate adsorb as monolayers with their alkyl chains either in the upright or tilted configuration, depending on their concentration. If large supramolecular structures were present in the solvent, i.e., for sorbitan monolaurate and glycerol monooleate, micelle adsorption and rearrangement on the surface and multilayer formation took place, respectively. A correlation between the adsorption rate constant and the coefficient of friction of the organic friction modifiers was revealed in these studies, with the coefficient of friction decreasing with an increase in the adsorption rate. [ABSTRACT FROM AUTHOR]

Traditional acoustic wave based sensing devices such as quartz crystal microbalance (QCM) rely on the thin films coated on the piezoelectric substrates as the sensing films for chemical and biological detection. This study demonstrates that significant sensitivity enhancement over traditional film based QCM (QCM-F) devices can be achieved by simply attaching a poly(methylmethacrylate) (PMMA) micropillar film onto a QCM substrate (QCM-P) by using a UV-curable glue. Humidity absorption measurements shows that the unique resonance occurred between the micropillars and the piezoelectric substrate improved the mass sensitivity of the QCM sensor by more than eight-fold. In addition, the newly developed QCM-P sensor and traditional QCM-F were utilized to detect bovine serum albumin (BSA) protein immobilization on PMMA surfaces. It was found that the glued QCM-P was capable of measuring BSA at a much lower concentration (200 nM) in comparison to QCM-F (1500 nM). The glue-based micropillar QCM device showed great potential for improving the sensitivity, simplifying the fabrication process, and reducing the cost of QCM sensors for various biosensing and chemical usages. [ABSTRACT FROM AUTHOR]

Terminal oxidases of aerobic respiratory chains catalyze the transfer of electrons from the respiratory substrate, cytochrome c or quinol, to O2 with the formation of two H2O molecules. There are two known families of these membrane oxidoreductases: heme-copper oxidase superfamily and bd-type oxidase family (cytochromes bd) found in prokaryotes only. The redox reaction catalyzed by these enzymes is coupled to the generation of proton motive force used by the cell to synthesize ATP and to perform other useful work. Due to the presence of the proton pump, heme-copper oxidases create the membrane potential with a greater energy efficiency than cytochromes bd. The latter, however, play an important physiological role that enables bacteria, including pathogenic ones, to survive and reproduce under adverse environmental conditions. This review discusses the features of organization and molecular mechanisms of functioning of terminal oxidases from these two families in the light of recent experimental data. [ABSTRACT FROM AUTHOR]

In this research, adsorption of Candida rugosa lipase enzyme (CRLE) onto activated carbon obtained from apple bark was carried out, and the thermodynamic parameters of adsorption process were investigated. The surface structural change of lipase enzyme and activated carbon was studied. The thermodynamic functions such as enthalpy, entropy, Gibbs free energy, and activation energy were investigated in their experimental work. The thermodynamic parameters of ΔG∗, Ea, ΔH∗, and ΔS∗ were calculated as − 75.56, 13.42, − 15.29 kJ mol−1, and 202.2 J mol−1 K−1 for CRLE adsorption, respectively. The experiment results showed that the adsorption process of CRLE on activated carbon is spontaneous and exothermic. The maximum adsorption capacity according to CRLE was 5.5 pH. The maximum adsorption capacity of active carbon was found to be 96.2 mg/g at pH 5.5, 309.5 K, and initial enzyme concentration of 5.0 × 10−3 M. The protein molecules at this point are very stable that is close to the isoelectric point of lipase enzyme. As a result, we can say that the activated carbon can be used as an effective adsorbent for the adsorption of CRLE. [ABSTRACT FROM AUTHOR]

In this research, the adsorption of bovine serum albümin (BSA) onto activated carbon (AC) obtained from apple bark was carried out and the thermodynamic parameters of adsorption process were investigated. Besides, the functions involved in BSA attachment were examined by adsorption experiments on retention capacities for BSA at 298 K, pH of 7, ionic strength of 5.10−2 M, and initial concentration of 5.10−2 g L−1, respectively. The bovine serum albumin (BSA) adsorption experiment onto activated carbon (AC) indicated that the highest adsorption yield was achieved at pH 5.5. The BSA molecules at pH 5.5 are very stable and that pH value is close to isoelectronic point of BSA. The surface structural change of BSA and activated carbon was studied before and after the experiment using scanning electron microscopy (SEM) analysis and Fourier Transform Infrared Spectroscopy (FTIR). By the way, the thermodynamic functions such as Gibbs free energy (ΔG∗), activation energy (Ea), activation entalphy (ΔH∗), and activation entropy (ΔS∗) were calculated as − 66.17, 37.73, − 29.09 kJ mol−1, and + 124.42 J mol−1 K−1 for bovine serum albumin (BSA) adsorption, respectively. The adsorption of the process was investigated using Eyring and Arrhenius equations and the adsorption kinetic of BSA on AC was found to be coherent with the pseudo-second-order model. [ABSTRACT FROM AUTHOR]

The rare stable isotope of hydrogen, deuterium, has fascinated researchers since its discovery in the 1930s. Subsequent large-scale production of deuterium oxide, commonly known as heavy water, became a starting point for further research. Deuterium exhibits unique physicochemical properties as well as having the strongest kinetic isotope effects among all other elements. Moreover, a broad variety of morphological and physiological changes have been observed in deuterium-treated cells and organisms, including changes in fundamental processes such as cell division or energy metabolism. Even though our understanding of such alterations is still insufficient, it is evident that some of them make growth in a deuterium-enriched environment a challenging task. There seems to be certain species-specific limits to their tolerance to heavy water, where some organisms are unable to grow in heavy water whilst others have no difficulties. Although the effects of deuterium on living organisms are, in general, negative, some of its applications are of great biotechnological potential, as is the case of stable isotope-labelled compounds or deuterated drugs. [ABSTRACT FROM AUTHOR]

Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11 kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11 kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25 kDa), salivary D7 secreted protein (22 kDa), and hypothetical protein (13 kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies. [ABSTRACT FROM AUTHOR]

Soil compaction is a major environmental problem that affects plant growth and development. In this study, to further our understanding of its negative effects on plant growth, we investigated the effects of soil compaction on the growth, mineral absorption, and activities of key respiratory enzymes in soybean seedlings. We found that moderate-level soil compaction increased the activities of pyruvate kinase and phosphofructokinase in soybean seedling roots, enhancing the accumulation of P, K, Mg, Ca, and other elements. These accumulated elements, particularly Ca, increased the number of fibrous upper roots, but reduced root length and inhibited plant growth. High-level soil compaction inhibited the accumulation of P, K, Mg, Mn, Fe, Cu, and Zn and increased the accumulation of Ca via decreasing the activities of isocitrate dehydrogenase and cytochrome c oxidase. These effects led to a decreased root cell size, blurred root cell boundaries, and the inhibition of plant growth. Taken together, our results provide a new insight into the mechanisms by which soil compaction inhibits plant growth. [ABSTRACT FROM AUTHOR]

Maintaining gradients of solvated protons and alkali metal ions such as Na+ and K+ across membranes is critical for cellular function. Over the last few decades, both the interactions of protons and alkali metal ions with phospholipid membranes have been studied extensively and the reported interactions of these ions with phospholipid headgroups are very similar, yet few studies have investigated the potential interdependence between proton and alkali metal ion binding at the water–lipid interface. In this short review, we discuss the similarities between the proton–membrane and alkali ion–membrane interactions. Such interactions include cation attraction to the phosphate and carbonyl oxygens of the phospholipid headgroups that form strong lipid–ion and lipid–ion–water complexes. We also propose potential mechanisms that may modulate the affinities of these cationic species to the water–phospholipid interfacial oxygen moieties. This review aims to highlight the potential interdependence between protons and alkali metal ions at the membrane surface and encourage a more nuanced understanding of the complex nature of these biologically relevant processes. [ABSTRACT FROM AUTHOR]

The scientific interest in gas sensors is continuously increasing because of their environmental, medical, industrial, and domestic applications. This has resulted in an increasing number of investigations being reported in the literature and communicated at conferences. The present review, organized in two parts, addresses the peculiarities of gas sensors based on mass-sensitive transducers, starting with their structure and functionality and progressing to implementation and specific use. In this first part of the review, we discuss the constructional peculiarities and operation regions and the physical and chemical processes governing the reception and transduction functions and the way in which they influence the sensor sensing parameters/features. Scientific outcomes and trends in research into gas sensors based on mass sensitive transducers are also considered. [ABSTRACT FROM AUTHOR]

In the paper we investigate the fractional-order evaluation of the fractional-order discrete integration element. We assume that the input and output signals are known. The main problem is to calculate fractional-order value. From a theoretical point of view there is no mathematical problem of the solution. One should solve linear algebraic equation or find roots of a polynomial in a variable ν. The problem arises when the measured output signal contains a noise. Then, the solution is unsettled because the polynomial roots are very sensitive to coefficients variability. In the paper we propose a method of evaluating of the discrete integrator fractional-order. The investigations are supported by numerical examples. [ABSTRACT FROM AUTHOR]

Pyogenic granuloma (PG) is a common benign vascular proliferation which often occurs on the head, neck, hands, and feet. Among the various treatment options for PG, surgical excision is the most effective treatment which offers the lowest overall recurrence rates and also provides the exact diagnosis. However, it could have difficulties to do the surgery when lesions are located on the fingers and toes, especially very near to the nails, so laser may be a very good alternative choice. In this article, we evaluated the clinical efficacy and safety of neodymium-yttrium aluminum garnet (Nd:YAG) laser for treatment of PG located on the fingers and toes. Twenty-one patients with 21 PGs located on the fingers and toes were treated by multispot Nd-YAG laser. We chose monopulse (pulse width 10.5-13.5 ms; energy 100-125 J/cm2); treatment interval was 3-4 weeks. All lesions disappeared after one or two treatments. There was no apparent scar formation, no impact on the function of the fingers and toes, no damage to nail growth, and no recurrence in more than 12-month follow-up. Nd-YAG is an effective and safety treatment option for treatment of PG located on the fingers and toes. [ABSTRACT FROM AUTHOR]

The CuA center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the CuA site (the TtCuA protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0-9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCuA increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the CuA site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCuA through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines. [ABSTRACT FROM AUTHOR]

The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer. [ABSTRACT FROM AUTHOR]

We have theoretically investigated the substituted effect on the first excited-state proton-transfer process of nX7AI-H2O (n = 2~6, X = F, Cl, Br) complex at the TD-M06-2X/6-31 + G(d, p) level. Here X is the substituted halogen atom, and n value denotes the substituted position of X, such as C2, C3, C4, C5, or C6. For the substituted 7-azaindole clusters, 6X7AI-H2O molecule is the most stable structure in water. The replacement of halogen atom X does not affect the characters of the HOMO and LUMO, but influence the S0 → S1 adiabatic transition energies of nX7AI-H2O (n = 2~6, X = F, Cl, Br). Our calculated results show that the double proton transfer occurs in a concerted but asynchronous protolysis pathway no matter which H atom is replaced by halogen atom. The halogen substitution changes the structural parameters evidently and leads to amply the asynchronousity during the proton-transfer process. The ESPT barrier height increases or decreases due to the halogen atom and substituted position. [ABSTRACT FROM AUTHOR]

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H+-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the KD-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters.ᅟ [ABSTRACT FROM AUTHOR]