Proton Translocation Research Articles

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :β3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :β3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑβγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits β and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal β-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

Evidence for the feasibility of transmembrane proton gradient regulating oxytetracycline extracellular biodegradation mediated by biosynthesized palladium nanoparticles

Extracellular biodegradation is a promising technology for removing antibiotics and repressing the spread of resistance genes, but the strategy is limited by the low extracellular electron transfer (EET) efficiency of microorganisms. In this work, biogenic Pd0 nanoparticles (bio-Pd0) were introduced in cells in situ to enhance oxytetracycline (OTC) extracellular degradation and the effects of transmembrane proton gradient (TPG) on EET and energy metabolism mediated by bio-Pd0 were investigated. The results indicated that the intracellular OTC concentration gradually decreased with increase in pH due to the simultaneous decreases of OTC adsorption and TPG-dependent OTC uptake. On the contrary, the efficiency of OTC biodegradation mediated by bio-Pd0@B. megaterium showed a pH-dependent increase. The negligible intracellular OTC degradation, the high dependence of OTC biodegradation on respiration chain and the results on enzyme activity and respiratory chain inhibition experiments showed that NADH-dependent (rather than FADH2-dependent) EET process mediated by substrate-level phosphorylation modulated OTC biodegradation due to high energy storage and proton translocation capacity. Moreover, the results showed that altering TPG is an efficient approach to improve EET efficiency, which can be attributed to the increased NADH generation by the TCA cycle, enhanced transmembrane electron output efficiency (as evidenced by increased intracellular electron transfer system (IETS) activity, the negative shift of onset potential, and enhanced one-electron transfer through bound flavin) and stimulation of substrate-level phosphorylation energy metabolism catalyzed by succinic thiokinase (STH) under low TPG conditions. The results of structural equation model that OTC biodegradation was directly and positively modulated by the net outward proton flux as well as STH activity, and indirectly regulated by TPG through NADH level and IETS activity confirmed the previous findings. This study provides a new perspective for engineering microbial EET and application of bioelectrochemistry processes in bioremediation.

Mirror proteorhodopsins

Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name “mirror proteorhodopsins”, from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest.

Photochemistry of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2 and Its Implications on Microbial Rhodopsin Research: Retrospective and Perspective.

The discovery of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) in 2013 has changed the paradigm that cation transport in microbial rhodopsins is restricted to the translocation of protons. Even though this finding is already remarkable by itself, it also reignited more general discussions about the functional mechanism of ion transport. The unique composition of the retinal binding pocket in KR2 with a tight interaction between the retinal Schiff base and its respective counterion D116 also has interesting implications on the photochemical pathway of the chromophore. Here, we discuss the most recent advances in our understanding of the KR2 functionality from the primary event of photon absorption by all-trans retinal up to the actual protein response in the later phases of the photocycle, mainly from the point of view of optical spectroscopy. In this context, we furthermore highlight some of the ongoing debates on the photochemistry of microbial rhodopsins and give some perspectives for promising future directions in this field of research.

Artificial organelles for sustainable chemical energy conversion and production in artificial cells: Artificial mitochondrion and chloroplasts.

Sustainable energy conversion modules are the main challenges for building complex reaction cascades in artificial cells. Recent advances in biotechnology have enabled this sustainable energy supply, especially the adenosine triphosphate (ATP), by mimicking the organelles, which are the core structures for energy conversion in living cells. Three components are mainly shared by the artificial organelles: the membrane compartment separating the inner and outer parts, membrane proteins for proton translocation, and the molecular rotary machine for ATP synthesis. Depending on the initiation factors, they are further categorized into artificial mitochondrion and artificial chloroplasts, which use chemical nutrients for oxidative phosphorylation and light for photosynthesis, respectively. In this review, we summarize the essential components needed for artificial organelles and then review the recent progress on two different artificial organelles. Recent strategies, purified and identified proteins, and working principles are discussed. With more study on the artificial mitochondrion and artificial chloroplasts, they are expected to be very powerful tools, allowing us to achieve complex cascading reactions in artificial cells, like the ones that happen in real cells.

Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase

F1Fo ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To further investigate the role of these residues and the chemical requirements for activity at these positions, we generated 13 substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and passive H+ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge or removal of polarity caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with cardiolipin.

The dual role of a multi-heme cytochrome in methanogenesis: MmcA is important for energy conservation and carbon metabolism in Methanosarcina acetivorans.

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c-type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (∆mmcA-rnf) or just the MmcA subunit (∆mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA is required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin oxidation that arise in each strain. Transcriptomic data suggest that the ∆mmcA mutant might oxidize ferredoxin by upregulating the cytosolic Wood-Ljundahl pathway for acetyl-CoA synthesis, whereas the ∆mmcA-rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to oxidize ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of rnf or mmcA leads to global transcriptional changes of genes involved in methanogenesis, carbon assimilation and regulation. Overall, our study provides systems-level insights into the non-overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA.

Exploring the transport cycle of DgoT, a bacterial homolog of human vesicular glutamate transporters.

The solute carrier 17 (SLC17) family belongs to the major facilitator superfamily (MFS) and encompasses vesicular glutamate transporters (VGLUTs), inorganic phosphate transporters, the lysosomal H+/sialic acid cotransporter sialin, and the vesicular nucleotide transporter (VNUT). SLC17 members mediate symport or exchange of various substrates with H+ at distinct stoichiometries. A bacterial D-galactonate transporter (DgoT) from E. coli shows high sequence similarity with mammalian VGLUTs and sialin. We used a combination of experimental and computational approaches to explore DgoT transport mechanism. In solid-supported membrane (SSM)-based electrophysiology, rapid galactonate application triggers a transient current generated by substrate binding and subsequent conformational changes, followed by a slower electrogenic transport. Transport current at different pH values demonstrates inhibition of H+ release and binding with apparent pK values of 6.7 and 9.2, respectively. We demonstrated that DgoT functions as galactonate/H+ symporter with 1:2 stoichiometry. There are four charged residues present in the membrane spanning region of DgoT, of which three (R47, R126 and E133) are conserved among SLC17 family. Mutations D46N, E133Q, R47Q and R126Q lead to loss of transport activity, while selective binding of galactonate remains intact. Analysis of pre-steady currents indicates that D46 and E133 are involved in proton translocation, while R47 and R126 are necessary for conformational changes. In all-atom molecular dynamics simulations, protonation of D46 and E133 leads to rearrangement of transmembrane (TM) helices TM1 and TM7 that gate access to the substrate-binding site from the extracellular side. Substrate binding then induces the closure of the periplasmic gate and transition to the inward-open conformation, where substrate is released, in agreement with the alternating access mechanism used by other MFS transporters. Based on our experimental and computational results, we propose a detailed transport model for galactonate/H+ cotransport in DgoT.

Pathological role of high sugar in mitochondrial respiratory chain defect - augmented mitochondrial stress.

Respiratory complex anomalies could lead to the production of ROS. Ca2+, at physiological levels serves as a second messenger in many physiological functions. However, a research group reported that mitochondrial calcium [Ca2+]m overload leads to ROS production which could be lethal to the mitochondria through various mechanisms. The F1F0-ATPase (ATP synthase or complex V) is the enzyme that is responsible for catalyzing the final step of oxidative phosphorylation. Mitochondrial complex V T8993G mutation specifically blocks the translocation of protons across the intermembrane space thereby blocking ATP synthesis leading to Neuropathy, Ataxia and Retinitis Pigmentosa Syndromes (NARP). The main aim undertaking this study is to explore the possibility of [Ca2+]m overload mediating the pathological roles of high glucose in defective respiratory chain mediated mitochondrial stress. The NARP cybrids are ordered and are established in vitro experimental models for the cells with F1FO-ATPase defects, these cells harbor 98% of mtDNA T8993G mutation. Their counterparts 143B osteosarcoma cell lines are the parental cell lines used for comparisons. We uncovered that, NARP cells mediated and enhanced the death of the cells (apoptosis) when incubated with hydrogen peroxide (H2O2) and high glucose respectively as depicted by the MTT assay of cell viability. Furthermore, using the fluorescence probe-coupled laser scanning confocal imaging microscopy, NARP cells significantly enabled mitochondrial reactive oxygen species (mROS) formation and enhanced the depolarization of the mitochondrial membrane potential (ΔΨm). Elucidating the mechanisms of sugar enhanced toxicity on mitochondria would help in the future to alleviate the symptoms of patients associated with Neuritis Ataxia Retinitis Pigmentosa (NARP) syndromes.

A Study on Mechanisms Underlying Proton Transport in Proton Pump-type Microbial Rhodopsins

Microbial rhodopsins are photoreceptive membrane proteins composed of seven transmembrane α-helical apoproteins (opsin) and a covalently bound retinal chromophore. Microbial rhodopsins exhibit a cyclic photochemical reaction referred to as photocycle when illuminated. During their photocycles, these proteins perform various functions such as ions transport and photosensing. Among the various functional types of rhodopsins found to date, we have focused on the utility of proton pump-type microbial rhodopsins as optogenetic tools for optical pH control in cells or organelles. To develop effective toolkits for this purpose, a deeper understanding of the proton-pumping mechanism in these rhodopsins may be required. In this review, we first introduce a useful experimental method for measuring rapid transient pH changes with photoinduced proton uptake/release using transparent tin oxide (SnO2) or indium-tin oxide (ITO) electrodes. In addition, we describe the unique pH-dependent behavior of the photoinduced proton transfer sequence as well as the vectoriality of proton transportation in proteorhodopsin (PR) from marine eubacteria. Through intensive ITO experiments over wide pH range, including extremely high or low pH values, in combination with photoelectric measurements using Xenopus oocytes or a thin polymer film "Lumirror," we encountered several interesting observations on photoinduced proton transfer in PR:1) proton uptake/release sequence reversal and potential proton translocation direction reversal under alkali conditions, and 2) fast proton release from D227, a secondary counterion of the protonated retinal Schiff base at acidic pH values.

CRISPR/Cas9-mediated gene editing of vacuolar ATPase subunit d mediates phytohormone biosynthesis and virus resistance in rice.

Vacuolar ATPases (V-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; OsV-ATPase subunit d (OsV-ATPase d) is part of an integral, membrane-embedded V0 complex in the V-ATPase complex. Whether OsV-ATPase d is involved in phytohormone biosynthesis and resistance in rice remains unknown. The knockout mutants of OsV-ATPase d in rice were generated using the CRISPR/Cas9 system, and mutation of OsV-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed that OsV-ATPase d is probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Compared to wild type, mutation of OsV-ATPase d significantly increased JA and ABA biosynthesis and resistance against Southern rice black-streaked dwarf virus (SRBSDV), but it decreased resistance against Rice stripe virus (RSV) in rice. The data presented in this study reveal that OsV-ATPase d mediates phytohormone biosynthesis and virus resistance in rice and can be selected as a potential target for resistance breeding in rice.

Cryo-EM structure of the respiratory I + III2 supercomplex from Arabidopsis thaliana at 2 Å resolution

Protein complexes of the mitochondrial respiratory chain assemble into respiratory supercomplexes. Here we present the high-resolution electron cryo-microscopy structure of the Arabidopsis respiratory supercomplex consisting of complex I and a complex III dimer, with a total of 68 protein subunits and numerous bound cofactors. A complex I-ferredoxin, subunit B14.7 and P9, a newly defined subunit of plant complex I, mediate supercomplex formation. The component complexes stabilize one another, enabling new detailed insights into their structure. We describe (1) an interrupted aqueous passage for proton translocation in the membrane arm of complex I; (2) a new coenzyme A within the carbonic anhydrase module of plant complex I defining a second catalytic centre; and (3) the water structure at the proton exit pathway of complex III2 with a co-purified ubiquinone in the QO site. We propose that the main role of the plant supercomplex is to stabilize its components in the membrane.

Road-blocker HSP disease mutation disrupts pre-organization for ATP hydrolysis in kinesin through a second sphere control

Kinesin motor proteins perform several essential cellular functions powered by the adenosine triphosphate (ATP) hydrolysis reaction. Several single-point mutations in the kinesin motor protein KIF5A have been implicated to hereditary spastic paraplegia disease (HSP), a lethal neurodegenerative disease in humans. In earlier studies, we have shown that a series of HSP-related mutations can impair the kinesin's long-distance displacement or processivity by modulating the order-disorder transition of the linker connecting the heads to the coiled coil. On the other hand, the reduction of kinesin's ATP hydrolysis reaction rate by a distal asparagine-to-serine mutation is also known to cause HSP disease. However, the molecular mechanism of the ATP hydrolysis reaction in kinesin by this distal mutation is still not fully understood. Using classical molecular dynamics simulations combined with quantum mechanics/molecular mechanics calculations, the pre-organization geometry required for optimal hydrolysis in kinesin motor bound to α/β-tubulin is determined. This optimal geometry has only a single salt-bridge (of the possible two) between Arg203-Glu236, putting a reactive water molecule at a perfect position for hydrolysis. Such geometry is also needed to create the appropriate configuration for proton translocation during ATP hydrolysis. The distal asparagine-to-serine mutation is found to disrupt this optimal geometry. Therefore, the current study along with our previous one demonstrates how two different effects on kinesin dynamics (processivity and ATP hydrolysis), caused by a different set of genotypes, can give rise to the same phenotype leading to HSP disease.

Analysis of Human Clinical Mutations of Mitochondrial ND1 in a Bacterial Model System for Complex I.

The most common causes of mitochondrial dysfunction and disease include mutations in subunits and assembly factors of Complex I. Numerous mutations in the mitochondrial gene ND1 have been identified in humans. Currently, a bacterial model system provides the only method for rapid construction and analysis of mutations in homologs of human ND1. In this report, we have identified nine mutations in human ND1 that are reported to be pathogenic and are located at subunit interfaces. Our hypothesis was that these mutations would disrupt Complex I assembly. Seventeen mutations were constructed in the homologous nuoH gene in an E. coli model system. In addition to the clinical mutations, alanine substitutions were constructed in order to distinguish between a deleterious effect from the introduction of the mutant residue and the loss of the original residue. The mutations were moved to an expression vector containing all thirteen genes of the E. coli nuo operon coding for Complex I. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity and proton translocation were measured. Samples were also tested for assembly by native gel electrophoresis and for expression of NuoH by immunoblotting. A range of outcomes was observed: Mutations at four of the sites allow normal assembly with moderate activity (50−76% of wild type). Mutations at the other sites disrupt assembly and/or activity, and in some cases the outcomes depend upon the amino acid introduced. In general, the outcomes are consistent with the proposed pathogenicity in humans.

Novel TCIRG1 Mutations Causing Congenital Neutropenia

Background: Severe congenital neutropenia (SCN) is a bone marrow failure disorder leading to severe neutropenia related infections and leukemia. More than 30 different genes associated with this disorder have been identified. We previously identified a novel heterozygous missense mutation (R736S) of TCIRG1 as the probable cause for SCN in a large multigenerational family through exome sequencing (Makaryan et al; Hum Mutat. 2014 Jul;35(7):824-7). We also identified a statistically significant correlation of rare missense variants of TCIRG1 with lower ANC in three cohorts from the NHLBI Grand Opportunity Exome Sequencing Project (GO ESP; Rosenthal et al; Genet Epidemiol. 2016 Sep;40(6):470-4). Recently, we have discovered a novel TCIRG1 variant (R736C) at the same position in a new family with congenital neutropenia. TCIRG1 encodes subunit a3, a 116-kD component (OC116) of V-ATPases, vacuolar proton pumps which mediate H+ transport through membranes. Homozygous loss-of-function mutations of TCIRG1 are the most common cause of autosomal recessive osteopetrosis (ARO). Mutations R736S and R736C occur at an amino acid residue critical for proton translocation. There are no reports in the literature of individuals with heterozygous mutations at this locus. This is in contrast to the numerous healthy individuals with various heterozygous variants throughout the TCIRG1 gene. Parents of ARO patients harboring homozygous TCIRG1 mutations are considered healthy with no musculoskeletal or hematopoietic abnormalities. Pathogenesis of TCIRG1-associated neutropenia is largely unknown. Hypothesis: We hypothesize that certain heterozygous TCIRG1 variants, like R736S and R736C, may cause SCN through specific molecular pathways. Methods: Family and medical history, differential WBC counts and whole exome sequencing/Identity by Decent (IBD) analysis were used to identify the genetic basis of neutropenia in the previously reported family. Sanger sequencing was used to confirm the findings of exome sequencing and to identify other affected individuals. The R736C mutation was identified by whole exome sequencing with further site-specific analysis of 35 genes. Affected relatives were identified by Sanger sequencing. The Polyphen 2 online prediction tool was used to predict the impact of both substitutions on TCIRG1 function. Results: Discovery and details of the original family were described in our previous report: (Makaryan et al; Hum Mutat. 2014 Jul;35(7):824-7). The second family was discovered in Taiwan, where the primary physician contacted SCNIR about a young patient with neutropenia who had a novel TCIRG1 mutation. Shortly thereafter, we identified two more affected family members. In both families the severity of recurrent bacterial infections is variable, roughly correlating with the severity of the neutropenia. For example, the grandmother of the index patient in the second family was not aware that she had neutropenia. The novel R736C TCIRG1 variant is located in the same position as the previous R736S variant. Arginine in this position is evolutionary highly conserved and both these substitutions cause a major change in amino acid size and properties. Polyphen-2 analysis predicts the effects of both mutations "probably damaging” (score 1.0). Conclusions: Understanding the genetic, epidemiological and molecular basis of TCIRG1-associated neutropenia is important for improving the diagnosis and treatment of this form of congenital neutropenia. The pathogenesis of TCIRG1-associated neutropenia is unknown. It is very important to emphasize that carriers of pathogenic variants from both families have neutropenia with variable severity. The report of this new family suggests that there may be other patients and families with neutropenia caused by mutations in TCIRG1. Population based genome studies, careful family histories and whole genome/exome sequencing will be highly valuable for identifying and helping new patients with this disorder.

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420–450 nm), alpha (560–630 nm) and red (630–700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨm) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨm-dependent, out of equilibrium, and therefore much slower than the ΔΨm-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.

Biomimetic Artificial Proton Channels.

One of the most common biochemical processes is the proton transfer through the cell membranes, having significant physiological functions in living organisms. The proton translocation mechanism has been extensively studied; however, mechanistic details of this transport are still needed. During the last decades, the field of artificial proton channels has been in continuous growth, and understanding the phenomena of how confined water and channel components mediate proton dynamics is very important. Thus, proton transfer continues to be an active area of experimental and theoretical investigations, and acquiring insights into the proton transfer mechanism is important as this enlightenment will provide direct applications in several fields. In this review, we present an overview of the development of various artificial proton channels, focusing mostly on their design, self-assembly behavior, proton transport activity performed on bilayer membranes, and comparison with protein proton channels. In the end, we discuss their potential applications as well as future development and perspectives.

On the role of ubiquinone in the proton translocation mechanism of respiratory complex I.

Complex I converts oxidoreduction energy into a proton electrochemical gradient across the inner mitochondrial or bacterial cell membrane. This gradient is the primary source of energy for aerobic synthesis of ATP. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) by ubiquinone (Q) yields NAD+ and ubiquinol (QH2 ), which is tightly coupled to translocation of four protons from the negatively to the positively charged side of the membrane. Electrons from NADH oxidation reach the iron-sulfur centre N2 positioned near the bottom of a tunnel that extends circa 30 Å from the membrane domain into the hydrophilic domain of the complex. The tunnel is occupied by ubiquinone, which can take a distal position near the N2 centre or proximal positions closer to the membrane. Here, we review important structural, kinetic and thermodynamic properties of ubiquinone that define its role in complex I function. We suggest that this function exceeds that of a mere substrate or electron acceptor and propose that ubiquinone may be the redox element of complex I coupling electron transfer to proton translocation.

F1FO ATP synthase molecular motor mechanisms.

The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of c-subunits that is attached to subunit-γ. This c-ring rotates relative to the subunit-a stator in response to transmembrane proton flow driven by a pH gradient, which drives subunit-γ rotation in the opposite direction to force ATP synthesis in F1. Single-molecule studies of F1FO embedded in lipid bilayer nanodisks showed that the c-ring transiently stopped F1-ATPase-driven rotation every 36° (at each c-subunit in the c10-ring of E. coli F1FO) and was able to rotate 11° in the direction of ATP synthesis. Protonation and deprotonation of the conserved carboxyl group on each c-subunit is facilitated by separate groups of subunit-a residues, which were determined to have different pKa’s. Mutations of any of any residue from either group changed both pKa values, which changed the occurrence of the 11° rotation proportionately. This supports a Grotthuss mechanism for proton translocation and indicates that proton translocation occurs during the 11° steps. This is consistent with a mechanism in which each 36° of rotation the c-ring during ATP synthesis involves a proton translocation-dependent 11° rotation of the c-ring, followed by a 25° rotation driven by electrostatic interaction of the negatively charged unprotonated carboxyl group to the positively charged essential arginine in subunit-a.

Hydroxide Ion Mechanism for Long-Range Proton Pumping in the Third Proton Transfer of Bacteriorhodopsin.

In bacteriorhodopsin, representative light-driven proton pump, five proton transfers yield vectorial active proton translocation, resulting in a proton gradient in microbes. Third proton transfer occurs from Asp96 to the Schiff base on the photocycle, which is expected to be a long-range proton transfer via the Grotthuss mechanism through internal water molecules. Here, large-scale quantum molecular dynamics simulations are performed for the third proton transfer, where all the atoms (∼50000 atoms) are treated quantum-mechanically. The simulations demonstrate that two reaction paths exist along the water wire, namely, via hydronium and via hydroxide ions. The free energy analysis confirms that the path via hydroxide ions is considerably favorable and consistent with the observed lifetime of the transient water wire. Therefore, the proposed hydroxide ion mechanism, as in the first proton transfer, is responsible for the third long-range proton transfer.