Stapled NRPS enhances the production of valinomycin in Escherichia coli.

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases-NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability.

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨm). Chemiosmosis requires ion exchangers to remove Na+, K+, Ca2+, PO43-, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K+, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H2O by osmosis. The effects of K+ uptake through ligand-specific mK+ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K+ influx is likely its effects on H+ cycling and bioenergetics facilitated by mitochondrial (m) K+/H+ exchange (mKHE), though the kinetics and consequences of K+ efflux by KHE are not well described. We hypothesized that a major role of K+ influx/efflux is stimulation of respiration via the influx of H+ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK+ cycle to capture changes in mitochondrial volume, pHm, ΔΨm, and respiration that would reflect a role for H+ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K+ buffer at pH 6.9, or in a 140 mM Cs+ buffer at pH 7.6 or 6.9 with added 10 mM K+, minimal Ca2+ and free of Na+. O2 content was measured by a Clark electrode, and pHm, ΔΨm, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpHm = pHin-pHext) at pHext 6.9> > 7.6, so that ΔΨm was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ ionophore valinomycin depolarized ΔΨm. We postulate that K+ efflux-induced H+ influx via KHE causes an inward H+ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpHm, the smaller component of the overall proton motive force, ΔµH+. Thus ΔpHm establishes and maintains the ΔΨm required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K+ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.

An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells.

INTRODUCTION: Commercially-available resazurin-based reagents used for cell viability assessment contain varying amounts of resorufin; these may contribute to differences in autofluorescence, signal-to-background (S/B) ratio and the dynamic range of the assay. OBJECTIVES: This in vitro study compares the sensitivity of a new, high-sensitivity PrestoBlue (hs-PB) assay with standard PrestoBlue (PB) in assessing the efficacy of valinomycin and antimycin A in human vascular endothelial EA.hy926 cells, as well as cell viability. METHODS: The metabolic activity of EA.hy926 was evaluated based on resorufin fluorescence (PB assays) or formazan absorbance (MTT assay). RESULTS: The hs-PB assay demonstrated lower resorufin autofluorescence than the PB, resulting in a ≥ 1.4-fold increase in S/B ratio in hs-PB compared to PB. Valinomycin was more potent cytotoxic agent than antimycin A. The hs-PB, PB and MTT produced similar IC50 values for valinomycin. Antimycin A showed significantly higher potency in the MTT than in the resazurin-based assays. The EA.hy926 cells demonstrated higher metabolic activity in the presence of the antimycin A solvent - DMSO. CONCLUSION: All the examined methods may be used interchangeably to analyze drug cytotoxicity. Any differences in drug cytotoxicity observed between the assays may be due to relatively low drug potency and/or the influence of solvent on metabolism of assay reagent. The hs-PB assay appears to more effectively detect cell viability and produce a stronger signal than its conventional counterpart.

Computational exploration of natural peptides targeting ACE2.

Interaction between the SARS-COV-2 (2019 novel coronavirus) spike protein and ACE2 receptors expressed on cellular surfaces initialises viral attachment and consequent infection. Blocking this interaction shows promise for blocking or ameliorating the virus' pathological effects on the body. By contrast to work focusing on the coronavirus, which has significant potential diversity through possible accumulation of mutations during transmission, targeting the conserved ACE2 protein expressed on human cells offers an attractive alternative route to developing pharmacological prophylactics against viral invasion. In this study, we screened a virtual database of natural peptides in silico, with ACE2 as the target, and performed structural analyses of the interface region in the SARS-COV-2 RBD/ACE2 complex. These analyses have identified 15 potentially effective compounds. Analyses of ACE2/polypeptide interactions suggest that these peptides can block viral invasion of cells by stably binding in the ACE2 active site pocket. Molecular simulation results for Complestatin and Valinomycin indicate that they may share this mechanism. The discovery of this probable binding mechanism provides a frame of reference for further optimization, and design of high affinity ACE2 inhibitors that could serve as leads for production of drugs with preventive and therapeutic effects against SARS-COV-2. Communicated by Ramaswamy H. Sarma.

Polymorphic Forms of Valinomycin Investigated by NMR Crystallography.

A dodecadepsipeptide valinomycin (VLM) has been most recently reported to be a potential anti-coronavirus drug that could be efficiently produced on a large scale. It is thus of importance to study solid-phase forms of VLM in order to be able to ensure its polymorphic purity in drug formulations. The previously available solid-state NMR (SSNMR) data are combined with the plane-wave DFT computations in the NMR crystallography framework. Structural/spectroscopical predictions (the PBE functional/GIPAW method) are obtained to characterize four polymorphs of VLM. Interactions which confer a conformational stability to VLM molecules in these crystalline forms are described in detail. The way how various structural factors affect the values of SSNMR parameters is thoroughly analyzed, and several SSNMR markers of the respective VLM polymorphs are identified. The markers are connected to hydrogen bonding effects upon the corresponding (13C/15N/1H) isotropic chemical shifts of (CO, Namid, Hamid, Hα) VLM backbone nuclei. These results are expected to be crucial for polymorph control of VLM and in probing its interactions in dosage forms.

Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch disease suppressiveness.

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.

Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots.

A molecular robot is a next-generation biochemical machine that imitates the actions of microorganisms. It is made of biomaterials such as DNA, proteins, and lipids. Three prerequisites have been proposed for the construction of such a robot: sensors, intelligence, and actuators. This Minireview focuses on recent research on synthetic ion channels and DNA computing technologies, which are viewed as potential candidate components of molecular robots. Synthetic ion channels, which are embedded in artificial cell membranes (lipid bilayers), sense ambient ions or chemicals and import them. These artificial sensors are useful components for molecular robots with bodies consisting of a lipid bilayer because they enable the interface between the inside and outside of the molecular robot to function as gates. After the signal molecules arrive inside the molecular robot, they can operate DNA logic gates, which perform computations. These functions will be integrated into the intelligence and sensor sections of molecular robots. Soon, these molecular machines will be able to be assembled to operate as a mass microrobot and play an active role in environmental monitoring and in vivo diagnosis or therapy.

Synergistic proinflammatory interactions of microbial toxins and structural components characteristic to moisture-damaged buildings.

Indoor exposure to microbes and their structural and metabolic compounds is notoriously complex. To study proinflammatory interactions between the multiple microbial agents, macrophages derived from human THP-1 monocytic cells were exposed to several concentrations of microbial toxins alone (emodin, enniatin B, physcion, sterigmatocystin, valinomycin) and in combination with microbial structural components (bacterial lipopolysaccharide [LPS] or fungal ß-glucan). While the expression of proinflammatory cytokines TNFα and IL-1ß to single toxins alone was modest, low-dose co-exposure with structural components increased the responses of emodin, enniatin B, and valinomycin synergistically, both at the mRNA and protein level, as measured by RT-qPCR and ELISA, respectively. Co-exposure of toxins and ß-glucan resulted in consistent synergistically increased expression of several inflammation-related genes, while some of the responses with LPS were also inhibitory. Co-exposure of toxins with either ß-glucan or LPS induced also mitochondrial damage and autophagocytosis. The results demonstrate that microbial toxins together with bacterial and fungal structural components characteristic to moisture-damaged buildings can have drastic synergistic proinflammatory interactions at low exposure levels.

Type II thioesterase improves heterologous biosynthesis of valinomycin in Escherichia coli.

Heterologous expression of secondary metabolite biosynthesis pathways in a surrogate host, e.g. Escherichia coli, has emerged in recent years as an effective way to produce complex natural products. The nonribosomal peptide (NRP) antibiotic valinomycin has been recombinantly produced in E. coli through reconstitution of its biosynthetic pathway from the native producer Streptomyces tsusimaensis. In this study, a discrete protein type II thioesterase (TEII) encoded in the valinomycin gene cluster was coexpressed in the valinomycin producing E. coli strain. Valinomycin titers were significantly improved from 0.5 (without TEII coexpression) to 3.3 mg L(-1), which demonstrates the reconstitutive function of TEII involved in NRP biosynthesis. Based on a flask scale fed-batch cultivation system, repeated feeding of the glucose polymer during the cultivation further increased cell density and valinomycin titer up to 55 (OD600) and 13 mg L(-1), respectively. This indicates scalable high cell density cultivation in a bioreactor for overproduction of valinomycin will be a potential and feasible approach. In this work we present an in vivo example to show that TEII plays a positive role in heterologous valinomycin production.

Automated identification of depsipeptide natural products by an informatic search algorithm.

Nonribosomal depsipeptides are a class of potent microbial natural products, which include several clinically approved pharmaceutical agents. Genome sequencing has revealed a large number of uninvestigated natural-product biosynthetic gene clusters. However, while novel informatic search methods to access these gene clusters have been developed to identify peptide natural products, depsipeptide detection has proven challenging. Herein, we present an improved version of our informatic search algorithm for natural products (iSNAP), which facilitates the detection of known and genetically predicted depsipeptides in complex microbial culture extracts. We validated this technology by identifying several depsipeptides from novel producers, and located a large number of novel depsipeptide gene clusters for future study. This approach highlights the value of chemoinformatic search methods for the discovery of genetically encoded metabolites by targeting specific areas of chemical space.

Making a match for Valinomycin: steroidal scaffolds in the design of electroneutral, electrogenic anion carriers.

The natural product Valinomycin is a well-known transmembrane cation carrier. Despite being uncharged, this molecule can extract potassium ions from water without counterions and ferry them through a membrane interior. Because it only transports positive ions, it is electrogenic, mediating a flow of charge across the membrane. Equivalent agents for anions would be valuable research tools and may have therapeutic applications, especially in the treatment of "channelopathies" such as cystic fibrosis. However, no such molecules have been found in nature. In this Account, we describe our research toward synthetic and rationally designed "anti-Valinomycins". As our core approach to this problem, we used the steroid nucleus, provided by cholic acid, as a scaffold for the assembly of anion receptors. By positioning H-bond donors on this framework, especially urea and thiourea groups in conformationally constrained axial positions, we created binding sites capable of exceptionally high affinities (up to 10(11) M(-1) for R4N(+)Cl(-) in chloroform). The extended hydrocarbon surface of the steroid helped to maintain compatibility with nonpolar media. When we tested these "cholapods" for chloride transport in vesicles, they provided the first evidence for electrogenic anion transport mediated by electroneutral organic carriers: in other words, they are the first authenticated anti-Valinomycins. They also proved active in live cells that we grew and assayed in an Ussing chamber. In subsequent work, we have shown that the cholapods can exhibit very high activities, with transport observed down to carrier/lipid ratios of 1:250,000. We also understand some of the effects of structure on the activity of these molecules. For example, in most cases, powerful transporters also act as powerful receptors. On the other hand, some modifications which favor binding do not promote transport. We gained functional advantages by cyclizing the cholapod architecture, which encloses the anion binding site. We could also simplify the structure without compromising function. A steroid-inspired trans-decalin framework has proved highly effective and may lead to agents with practical advantages. Changing an ester side-chain in this system revealed a surprising effect, whereby increased length and/or lipophilicity resulted in substantially raised activity. Although much remains to be discovered about these anionophores, their high activities and intrinsic tuneabilities bode well for applications. In future work, we plan to develop and exploit these molecules as tools for biophysical research and to explore the possibility of useful biological activity.

Thapsigargin-induced endoplasmic reticulum stress is not accompanied by mitochondrial membrane potential dissipation in murine pro-B cells.

UNLABELLED: There are almost no data concerning the involvement of endoplasmic reticulum stress (Ca2+ fluxes) in the apoptosis of the pro-B cell type Ba/F3. Thus, we aimed the characterization of thapsigargin-induced effects on Ba/F3 cells in vitro. MATERIAL AND METHOD: For some experiments Ba/F3 cells were treated with 1 microM thapsigargin for 24 hours. To compare, we used as positive control the effects of valinomycin 10 microM. The negative control Ba/F3 cells received no treatment for 24 hours. After that, all batches of Ba/F3 cells were incubated in the presence of 1 mimcroM JC-1 (Sigma-Aldrich) at 37 degrees C for 30 minutes. RESULTS: From the normal Ba/F3 JC-1-control cells (20.000 events gated by flow cytometry) 77.61 +/- 2.90% are associating high and 22.39 = 2.90% lower mitochondrial membrane potential. In the case of thapsigargin, 75.49 +/- 1.78% of the Ba/F3 cells are associating high and 24.51 +/- 1.78% lower mitochondrial membrane potential. CONCLUSIONS: While mitochondrial permeability transition pore (MPTP) opening necessarily correlates with a loss of mitochondrial membrane potential (Psi(mt)), a decrease in Psi(mt) does not necessarily indicate MPTP opening. Also, endoplasmic reticulum stress regulation of high cytosolic calcium levels by thapsigargin may prompt the opening of the MPTP without necessarily triggering irreversible pore activation or subsequent apoptogenic factors in Ba/F3 cells.

Thiourea isosteres as anion receptors and transmembrane transporters.

Compounds containing cyanoguanidine and 3-amino-1,2,4-benzothiadiazine-1,1-dioxide have been studied as anion receptors and transporters. Significant affinity for oxo-anions was observed in organic solution and the receptors were found to function as transmembrane chloride/nitrate antiporters with transport rates enhanced in the presence of valinomycin-K(+) complex.

The role of bulk protein in local models of ion-binding to proteins: comparative study of KcsA, its semisynthetic analog with a locked-in binding site, and valinomycin.

In studying ion-selectivity in biomaterials, it is common to study ion-protein interactions within a local neighborhood around the ion. This local system analysis for the S(2) site of KcsA, its semisynthetic analog, and valinomycin yields the free energy change in exchanging K(+) with Na(+) in quantitative agreement with the value obtained by considering ion-interactions with the entire system. But the energetics of ion binding in the local system and in the entire system differ significantly and lead to different conclusions regarding the physical basis of ion selectivity. For configurations sampled from an all-atom simulation, we show that the selectivity free energy can be decomposed into a contribution arising from interactions of the ion with its local neighborhood, ΔW(local), and a term arising from the field imposed on the ion and the binding site by the rest of the medium, ΔW(ϕ). The local contribution ΔW(local) is numerically close to the actual free energy difference because the field contribution is small. The field contribution is small because of cancellation of inversely related ion-medium and site-medium interactions. Our analysis presents a rigorous foundation for the numerical success of the local system analysis and shows that its implications do not always hold for the entire protein.

Oxalate efflux transporter from the brown rot fungus Fomitopsis palustris.

An oxalate-fermenting brown rot fungus, Fomitopsis palustris, secretes large amounts of oxalic acid during wood decay. Secretion of oxalic acid is indispensable for the degradation of wood cell walls, but almost nothing is known about the transport mechanism by which oxalic acid is secreted from F. palustris hyphal cells. We characterized the mechanism for oxalate transport using membrane vesicles of F. palustris. Oxalate transport in F. palustris was ATP dependent and was strongly inhibited by several inhibitors, such as valinomycin and NH(4)(+), suggesting the presence of a secondary oxalate transporter in this fungus. We then isolated a cDNA, FpOAR (Fomitopsis palustris oxalic acid resistance), from F. palustris by functional screening of yeast transformants with cDNAs grown on oxalic acid-containing plates. FpOAR is predicted to be a membrane protein that possesses six transmembrane domains but shows no similarity with known oxalate transporters. The yeast transformant possessing FpOAR (FpOAR-transformant) acquired resistance to oxalic acid and contained less oxalate than the control transformant. Biochemical analyses using membrane vesicles of the FpOAR-transformant showed that the oxalate transport property of FpOAR was consistent with that observed in membrane vesicles of F. palustris. The quantity of FpOAR transcripts was correlated with increasing oxalic acid accumulation in the culture medium and was induced when exogenous oxalate was added to the medium. These results strongly suggest that FpOAR plays an important role in wood decay by acting as a secondary transporter responsible for secretion of oxalate by F. palustris.

Electrostatics of deformable lipid membranes.

It was recently demonstrated that significant local deformations of biological membranes take place due to the fields of charged peptides and ions, challenging the standard model of membrane electrostatics. The ability of ions to retain their immediate hydration environment, combined with the lack of sensitivity of permeability to ion type or even ion pairs, led us to question the extent to which hydration energetics and electrostatics control membrane ion permeation. Using the arginine analog methyl-guanidinium as a test case, we find that although hydrocarbon electronic polarizability causes dramatic changes in ion solvation free energy, as well as a significant change (approximately 0.4 V) in the membrane dipole potential, little change in membrane permeation energetics occurs. We attribute this to compensation of solvation terms from polar and polarizable nonpolar components within the membrane, and explain why the dipole potential is not fully sensed in terms of the locally deformed bilayer interface. Our descriptions provide a deeper understanding of the translocation process and allow predictions for poly-ions, ion pairs, charged lipids, and lipid flip-flop. We also report simulations of large hydrophobic-ion-like membrane defects and the ionophore valinomycin, which exhibit little membrane deformation, as well as hydrophilic defects and the ion channel gramicidin A, to provide parallels to membranes deformed by unassisted ion permeation.

Vesicular inhibitory amino acid transporter is a Cl-/gamma-aminobutyrate Co-transporter.

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of gamma-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Deltapsi) (positive inside) but not DeltapH. VIAAT-mediated GABA uptake had an absolute requirement for Cl(-) and actually accompanied Cl(-) movement. Kinetic analysis indicated that one GABA molecule and two Cl(-) equivalents were transported during one transport cycle. VIAAT in which Glu(213) was specifically mutated to alanine completely lost the ability to take up both GABA and Cl(-). Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl(-) transporter that co-transports Cl(-) with GABA or glycine in a Deltapsi dependent manner. It is concluded that Cl(-) plays an essential role in vesicular storage of GABA and glycine.

Valinomycin biosynthetic gene cluster in Streptomyces: conservation, ecology and evolution.

Many Streptomyces strains are known to produce valinomycin (VLM) antibiotic and the VLM biosynthetic gene cluster (vlm) has been characterized in two independent isolates. Here we report the phylogenetic relationships of these strains using both parsimony and likelihood methods, and discuss whether the vlm gene cluster shows evidence of horizontal transmission common in natural product biosynthetic genes. Eight Streptomyces strains from around the world were obtained and sequenced for three regions of the two large nonribosomal peptide synthetase genes (vlm1 and vlm2) involved in VLM biosynthesis. The DNA sequences representing the vlm gene cluster are highly conserved among all eight environmental strains. The geographic distribution pattern of these strains and the strict congruence between the trees of the two vlm genes and the housekeeping genes, 16S rDNA and trpB, suggest vertical transmission of the vlm gene cluster in Streptomyces with no evidence of horizontal gene transfer. We also explored the relationship of the sequence of vlm genes to that of the cereulide biosynthetic genes (ces) found in Bacillus cereus and found them highly divergent from each other at DNA level (genetic distance values >or= 95.6%). It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently.

Impedance analysis of valinomycin activity in nano-BLMs.

Nano-black lipid membranes (nano-BLMs) were obtained by functionalization of highly ordered porous alumina substrates with an average pore diameter of 60nm based on a self-assembled alkanethiol submonolayer followed by spreading of 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane on the hydrophobic substrate. By means of impedance spectroscopy, we analyzed the influence of the self-assembled alkanethiol submonolayer on the electrical properties of the nano-BLMs as well as their long-term stability. We were able to stably integrate nano-BLMs into a flow through system, which allowed us to readily exchange buffer solutions several times and accounts for mass transport phenomena. The ionophore valinomycin was successfully inserted into nano-BLMs and its transport activity monitored as a function of different potassium and sodium ion concentrations reflecting the specificity of valinomycin for potassium ions.

Deltapsi and DeltapH are equivalent driving forces for proton transport through isolated F(0) complexes of ATP synthases.

The membrane-embedded F(0) part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F(0) complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F(0) part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Deltapsi or DeltapH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F(1)F(0) holoenzyme, no significant difference was observed in the efficiency of DeltapH or Deltapsi as driving forces for H(+)-transport through F(0). Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H(+)/(s x F(0)) at Deltapsi of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H(+)-transport at initial rates of 6300 H(+)/(s x F(0)) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.