Transmembrane Electrochemical Gradient Research Articles

The fluoride exporter (CsFEX) regulates fluoride uptake/accumulation in Camellia sinensis under different pH

Fluoride (F) can be absorbed from the environment and hyperaccumulate in leaves of Camellia sinensis without exhibiting any toxic symptoms. Fluoride exporter in C. sinensis (CsFEX) could transport F to extracellular environment to alleviate F accumulation and F toxicity, but its functional mechanism remains unclear. Here, combining with pH condition of C. sinensis growth, the characteristics of CsFEX and mechanism of F detoxification were further explored. The results showed that F accumulation was influenced by various pH, and pH 4.5 and 6.5 had a greater impact on the F accumulation of C. sinensis. Through Non-invasive Micro-test Technology (NMT) detection, it was found that F uptake/accumulation of C. sinensis and Arabidopsis thaliana might be affected by pH through changing the transmembrane electrochemical proton gradient of roots. Furthermore, diverse expression patterns of CsFEX were induced by F treatment under different pH, which was basically up-regulated in response to high F accumulation, indicating that CsFEX was likely to participate in the process of F accumulation in C. sinensis and this process might be regulated by pH. Additionally, CsFEX functioned in the mitigation of F sensitivity and accumulation strengthened by lower pH in Escherichia coli and A. thaliana. Moreover, the changes of H+ flux and potential gradient caused by F were relieved as well in transgenic lines, also suggesting that CsFEX might play an important role in the process of F accumulation. Above all, F uptake/accumulation were alleviated in E. coli and A. thaliana by CsFEX through exporting F-, especially at lower pH, implying that CsFEX might regulate F accumulation in C. sinensis.

Nickel nitride-mediated nitric oxide generation for combating implant-associated infections

NiTi alloys are widely used in the fabrication of biomedical implants, and it is essential to endow them with selective antibacterial properties to avoid implant-associated infection. In this study, nickel nitride (Ni3N), as a promising new inorganic nitric oxide (NO) donor, was in situ constructed on the surface of NiTi alloys through a straightforward two-step plasma immersion ion implantation (PIII) strategy. Nickel sites were first exposed by physical bombardment with argon or metal plasma, followed by reaction with nitrogen plasma to form Ni3N. Ni3N can disrupt the transmembrane proton electrochemical gradient by capturing protons to produce ammonia species (NHx), resulting in the accumulation of reactive oxygen species (ROS) as a result of bacterial metabolic disruption. On the other hand, the generated NHx can further react with ROS to produce NO and reactive nitrogen species (RNS). As a result, the Ni3N films exhibited more than 99 % antibacterial efficiency against both gram-negative and gram-positive bacteria. They also demonstrated effective antibacterial properties in a rat subcutaneous implant model. In addition, the modified NiTi alloys exhibit good corrosion resistance and cytocompatibility. This study provides a green, safe, and inexpensive method for constructing metal nitride films on implant surfaces and offers new ideas for the clinical design and fabrication of novel antibacterial implants.

Fungal Plasma Membrane H+-ATPase: Structure, Mechanism, and Drug Discovery.

The fungal plasma membrane H+-ATPase (Pma1) pumps protons out of the cell to maintain the transmembrane electrochemical gradient and membrane potential. As an essential P-type ATPase uniquely found in fungi and plants, Pma1 is an attractive antifungal drug target. Two recent Cryo-EM studies on Pma1 have revealed its hexameric architecture, autoinhibitory and activation mechanisms, and proton transport mechanism. These structures provide new perspectives for the development of antifungal drugs targeting Pma1. In this article, we review the history of Pma1 structure determination, the latest structural insights into Pma1, and drug discoveries targeting Pma1.

Ion and lipid orchestration of secondary active transport.

Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondaryactive transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.

Experimental Research on the Influence of Ion Channels on the Healing of Skin Wounds in Rats

At the level of skin wounds, an electrical potential difference develops between the edges of the wound and the center of the wound, which favors the migration of cells in the process of their healing. Cells migrate in an electric field because they have a certain electrical membrane potential. This potential is due to differences in the transmembrane electrochemical gradient. The transmembrane electrochemical gradient is due to the migration of sodium, potassium, and calcium ions into the corresponding ion channels. If this is the case, the modification of the functionality of these ion channels should influence the membrane potential and, as a consequence, the wound healing process. In this experiment, we set out to investigate whether the chemical manipulation of ion channels by amiodarone influences the wound healing process. Amiodarone blocks several types of ion channels, but at different concentrations: at low concentrations, it blocks only potassium channels; at medium concentrations, potassium and calcium channels; and at high concentrations, it blocks potassium, calcium, and sodium channels. We worked on rats that were given experimental skin lesions and evaluated the influence of the healing of these lesions upon the topical administration of amiodarone in three concentrations, 200 nM, 2000 nM and 200,000 nM, compared to an untreated group and a group treated with benzyl alcohol, the amiodarone solvent. In our experimental conditions, low concentration amiodarone promoted wound healing both in terms of duration of healing and also in terms of speed of healing. This means that blocking some ions, possibly potassium channels, might promote wound healing.

A simple assay for inhibitors of mycobacterial oxidative phosphorylation

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and adenosine triphosphate (ATP) synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and non-specific membrane uncoupling. In this assay, IMVs from M. smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Non-specific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2 while thioridazine uncouples the mycobacterial membrane non-specifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

The state of mitochondrial membrane potential in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

Introduction. Mitochondria provide energy homeostasis of the cell by maintaining an optimal transmembrane electrochemical gradient (ΔΨm), which does not allow excessive formation of reactive oxygen species (ROS). However, under conditions of pathology, the normal functioning of mitochondria is disrupted, which can lead to ATP deficiency and/or increased production of ROS.Aim. The aim of this study was to investigate the ΔΨm parameters and their relationship with the expression of TRP channels in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease (COPD).Materials and methods. The study included 23 patients with COPD of varying severity, 8 smokers without signs of bronchial obstruction and 9 healthy volunteers who had never smoked. All subjects underwent spirometry to assess the lung function. ΔΨm was determined by staining the cells with tetramethylrhodamine ethyl ester (TMRE) and measuring the fluorescent signal by flow cytometry, under basal conditions and pro-inflammatory stimulation with phorbol-12-myristate-13-acetate (PMA).Results. We found that COPD patients were characterized by a significant increase in basal ΔΨm of monocytes (161.8 [153.8; 206.8] vs. 129.3 [75.5; 161.8], p=0.03) and lymphocytes (209,7 [184.7; 257.8] vs. 122.5 [67.9; 164.3], p=0.003) as compared with the control group. Stimulation of cells with PMA led to multidirectional changes in ΔΨm, while its increased level was still preserved in COPD. In monocytes of COPD patients, a decrease in ΔΨm in response to PMA stimulation was prevalent (75%), while in the majority (53.9%) of individuals in the control group ΔΨm, on the contrary, increased (p=0.08). In addition, among COPD patients, an increase in ΔΨm in monocytes was accompanied by an enhanced expression of TRPV4, while in the control group, among individuals with positive dynamics of ΔΨm, TRPV4 expression was, on the contrary, reduced.Conclusion. The increased level of ΔΨm in the mononuclears of COPD patients is consistent with previously detected enhanced ROS production, but does not support the assumption about energy deficit in the cells. The revealed differences in the relationship between TRPV4 expression and ΔΨm dynamics may indicate the presence of pathological features in TRP signaling in COPD patients.

ATP1A1-linked diseases require a malfunctioning protein product from one allele

Heterozygous germline variants in ATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αβ) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+ and K+ that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1+/− knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1+/− mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1-related diseases despite carrying a plasma-membrane protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

ATP yield of plant respiration: potential, actual and unknown.

The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

Modeling control and transduction of electrochemical gradients in acid-stressed bacteria

Transmembrane electrochemical gradients drive solute uptake and constitute a substantial fraction of the cellular energy pool in bacteria. These gradients act not only as "homeostatic contributors," but also play a dynamic and keystone role in several bacterial functions, including sensing, stress response, and metabolism. At the system level, multiple gradients interact with ion transporters and bacterial behavior in a complex, rapid, and emergent manner; consequently, experiments alone cannot untangle their interdependencies. Electrochemical gradient modeling provides a general framework to understand these interactions and their underlying mechanisms. We quantify the generation, maintenance, and interactions of electrical, proton, and potassium potential gradients under lactic acid-stress and lactic acid fermentation. Further, we elucidate a gradient-mediated mechanism for intracellular pH sensing and stress response. We demonstrate that this gradient model can yield insights on the energetic limitations of membrane transport, and can predict bacterial behavior across changing environments.

F1·Fo ATP Synthase/ATPase: Contemporary View on Unidirectional Catalysis

F1·Fo-ATP synthases/ATPases (F1·Fo) are molecular machines that couple either ATP synthesis from ADP and phosphate or ATP hydrolysis to the consumption or production of a transmembrane electrochemical gradient of protons. Currently, in view of the spread of drug-resistant disease-causing strains, there is an increasing interest in F1·Fo as new targets for antimicrobial drugs, in particular, anti-tuberculosis drugs, and inhibitors of these membrane proteins are being considered in this capacity. However, the specific drug search is hampered by the complex mechanism of regulation of F1·Fo in bacteria, in particular, in mycobacteria: the enzyme efficiently synthesizes ATP, but is not capable of ATP hydrolysis. In this review, we consider the current state of the problem of "unidirectional" F1·Fo catalysis found in a wide range of bacterial F1·Fo and enzymes from other organisms, the understanding of which will be useful for developing a strategy for the search for new drugs that selectively disrupt the energy production of bacterial cells.

Disease mutations of human α3 Na+/K+-ATPase define extracellular Na+ binding/occlusion kinetics at ion binding site III.

Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+, is central to the pathogenesis of neurological diseases such as alternating hemiplegia of childhood. Although Na+/K+-ATPase has 3 distinct ion binding sites I-III, the difficulty of distinguishing ion binding events at each site from the others hinders kinetic study of these transitions. Here we show that binding of Na+ at each site in the human α3 Na+/K+-ATPase can be resolved using extracellular Na+-mediated transient currents. When Na+/K+-ATPase is constrained to bind and release only Na+, three kinetic components: fast, medium, and slow, can be isolated, presumably corresponding to the protein dynamics associated with the binding (or release depending on the voltage step direction) and the occlusion (or deocclusion) of each of the 3 Na+. Patient-derived mutations of residues which coordinate Na+ at site III exclusively impact the slow component, demonstrating that site III is crucial for deocclusion and release of the first Na+ into the extracellular milieu. These results advance understanding of Na+/K+-ATPase mutation pathogenesis and provide a foundation for study of individual ions’ binding kinetics.

How to tune the absorption spectrum of chlorophylls to enable better use of the available solar spectrum

Photon capture by chlorophylls and other chromophores in light-harvesting complexes and photosystems is the driving force behind the light reactions of photosynthesis. Excitation of photosystem II allows it to receive electrons from the water-oxidizing oxygen-evolution complex and to transfer them to an electron-transport chain that generates a transmembrane electrochemical gradient and ultimately reduces plastocyanin, which donates its electron to photosystem I. Subsequently, excitation of photosystem I leads to electron transfer to a ferredoxin which can either reduce plastocyanin again (in so-called “cyclical electron-flow”) and release energy for the maintenance of the electrochemical gradient, or reduce NADP+ to NADPH. Although photons in the far-red (700–750 nm) portion of the solar spectrum carry enough energy to enable the functioning of the photosynthetic electron-transfer chain, most extant photosystems cannot usually take advantage of them due to only absorbing light with shorter wavelengths. In this work, we used computational methods to characterize the spectral and redox properties of 49 chlorophyll derivatives, with the aim of finding suitable candidates for incorporation into synthetic organisms with increased ability to use far-red photons. The data offer a simple and elegant explanation for the evolutionary selection of chlorophylls a, b, c, and d among all easily-synthesized singly-substituted chlorophylls, and identified one novel candidate (2,12-diformyl chlorophyll a) with an absorption peak shifted 79 nm into the far-red (relative to chlorophyll a) with redox characteristics fully suitable to its possible incorporation into photosystem I (though not photosystem II). chlorophyll d is shown by our data to be the most suitable candidate for incorporation into far-red utilizing photosystem II, and several candidates were found with red-shifted Soret bands that allow the capture of larger amounts of blue and green light by light harvesting complexes.

Tools for analyzing protonation states and for tracing proton transfer pathways with examples from the Rb. sphaeroides photosynthetic reaction centers.

Protons participate in many reactions. In proteins, protons need paths to move in and out of buried active sites. The vectorial movement of protons coupled to electron transfer reactions establishes the transmembrane electrochemical gradient used for many reactions, including ATP synthesis. Protons move through hydrogen bonded chains of waters and hydroxy side chains via the Grotthuss mechanism and by proton binding and release from acidic and basic residues. MCCE analysis shows that proteins exist in a large number of protonation states. Knowledge of the equilibrium ensemble can provide a rational basis for setting protonation states in simulations that fix them, such as molecular dynamics (MD). The proton path into the QB site in the bacterial reaction centers (RCs) of Rb. sphaeroides is analyzed by MD to provide an example of the benefits of using protonation states found by the MCCE program. A tangled web of side chains and waters link the cytoplasm to QB. MCCE analysis of snapshots from multiple trajectories shows that changing the input protonation state of a residue in MD biases the trajectory shifting the proton affinity of that residue. However, the proton affinity of some residues is more sensitive to the input structure. The proton transfer networks derived from different trajectories are quite robust. There are some changes in connectivity that are largely restricted to the specific residues whose protonation state is changed. Trajectories with QB•- are compared with earlier results obtained with QB [Wei et. al Photosynthesis Researchvolume152,pages153-165 (2022)] showing only modest changes. While introducing new methods the study highlights the difficulty of establishing the connections between protein conformation.

Modulation of the H+/ATP coupling ratio by ADP and ATP as a possible regulatory feature in the F-type ATP synthases

F-type ATP synthases are transmembrane enzymes, which play a central role in the metabolism of all aerobic and photosynthetic cells and organisms, being the major source of their ATP synthesis. Catalysis occurs via a rotary mechanism, in which the free energy of a transmembrane electrochemical ion gradient is converted into the free energy of ATP phosphorylation from ADP and Pi, and vice versa. An ADP, tightly bound to one of the three catalytic sites on the stator head, is associated with catalysis inhibition, which is relieved by the transmembrane proton gradient and by ATP. By preventing wasteful ATP hydrolysis in times of low osmotic energy and low ATP/ADP ratio, such inhibition constitutes a classical regulatory feedback effect, likely to be an integral component of in vivo regulation. The present miniview focuses on an additional putative regulatory phenomenon, which has drawn so far little attention, consisting in a substrate-induced tuning of the H+/ATP coupling ratio during catalysis, which might represent an additional key to energy homeostasis in the cell. Experimental pieces of evidence in support of such a phenomenon are reviewed.

Disease mutations of human α3 Na+/K+-ATPase define extracellular Na+ binding/occlusion kinetics at ion binding site III.

Na+/K+-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na+ for 2 K+, is central to the pathogenesis of neurological diseases such as alternating hemiplegia of childhood. Although Na+/K+-ATPase has 3 distinct ion binding sites I-III, the difficulty of distinguishing ion binding events at each site from the others hinders kinetic study of these transitions. Here, we show that binding of Na+ at each site in the human α3 Na+/K+-ATPase can be resolved using extracellular Na+-mediated transient currents. When Na+/K+-ATPase is constrained to bind and release only Na+, three kinetic components: fast, medium, and slow, can be isolated, presumably corresponding to the protein dynamics associated with the binding (or release depending on the voltage step direction) and the occlusion (or deocclusion) of each of the 3 Na+. Patient-derived mutations of residues which coordinate Na+ at site III exclusively impact the slow component, demonstrating that site III is crucial for deocclusion and release of the first Na+ into the extracellular milieu. These results advance understanding of Na+/K+-ATPase mutation pathogenesis and provide a foundation for study of individual ions' binding kinetics.

Cation-Chloride Cotransporters, Na/K Pump, and Channels in Cell Water/Ionic Balance Regulation Under Hyperosmolar Conditions: In Silico and Experimental Studies of Opposite RVI and AVD Responses of U937 Cells to Hyperosmolar Media.

Studying the transport of monovalent ions across the cell membrane in living cells is complicated by the strong interdependence of fluxes through parallel pathways and requires therefore computational analysis of the entire electrochemical system of the cell. Current paper shows how to calculate changes in the cell water balance and ion fluxes caused by changes in the membrane channels and transporters during a normal regulatory increase in cell volume in response to osmotic cell shrinkage (RVI) followed by a decrease in cell volume associated with apoptosis (AVD). Our recently developed software is used as a computational analysis tool and the established human lymphoid cells U937 are taken as an example of proliferating animal cells. It is found that, in contrast to countless statements in the literature that cell volume restoration requires the activation of certain ion channels and transporters, the cellular responses such as RVI and AVD can occur in an electrochemical system like U937 cells without any changes in the state of membrane channels or transporters. These responses depend on the types of chloride cotransporters in the membrane and differ in a hyperosmolar medium with additional sucrose and in a medium with additional NaCl. This finding is essential for the identification of the true changes in membrane channels and transporters responsible for RVI and AVD in living cells. It is determined which changes in membrane parameters predicted by computational analysis are consistent with experimental data obtained on living human lymphoid cells U937, Jurkat, and K562 and which are not. An essential part of the results is the developed software that allows researchers without programming experience to calculate the fluxes of monovalent ions via the main transmembrane pathways and electrochemical gradients that move ions across the membrane. The software is available for download. It is useful for studying the functional expression of the channels and transporters in living cells and understanding how the cell electrochemical system works.

Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3− Cotransporter NBCe1

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+–2HCO3− stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3− influx. However, NBCe1 in the kidney mediates HCO3− efflux (HCO3− reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3− efflux) than in murine colons (in which NBCe1 mediates HCO3− influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

Electron Transfer Coupled to Conformational Dynamics in Cell Respiration.

Cellular respiration is a fundamental process required for energy production in many organisms. The terminal electron transfer complex in mitochondrial and many bacterial respiratory chains is cytochrome c oxidase (CcO). This converts the energy released in the cytochrome c/oxygen redox reaction into a transmembrane proton electrochemical gradient that is used subsequently to power ATP synthesis. Despite detailed knowledge of electron and proton transfer paths, a central question remains as to whether the coupling between electron and proton transfer in mammalian mitochondrial forms of CcO is mechanistically equivalent to its bacterial counterparts. Here, we focus on the conserved span between H376 and G384 of transmembrane helix (TMH) X of subunit I. This conformationally-dynamic section has been suggested to link the redox activity with the putative H pathway of proton transfer in mammalian CcO. The two helix X mutants, Val380Met (V380M) and Gly384Asp (G384D), generated in the genetically-tractable yeast CcO, resulted in a respiratory-deficient phenotype caused by the inhibition of intra-protein electron transfer and CcO turnover. Molecular aspects of these variants were studied by long timescale atomistic molecular dynamics simulations performed on wild-type and mutant bovine and yeast CcOs. We identified redox- and mutation-state dependent conformational changes in this span of TMH X of bovine and yeast CcOs which strongly suggests that this dynamic module plays a key role in optimizing intra-protein electron transfers.

Structure-based insights into evolution of rhodopsins

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.