Proton Release Research Articles

Cations facilitate sulfidation of zero-valent iron by elemental sulfur: Mechanism and dechlorination application

The solid-solid reaction of microscale zero-valent iron (mZVI) with elemental sulfur (S0) in water can form sulfidated mZVI (S-mZVI) with high reactivity and selectivity. However, the inherent passivation layer of mZVI hinders the sulfidation. In this study, we demonstrate that ionic solutions of Me-chloride (Me: Mg2+, Ca2+, K+, Na+ and Fe2+) can accelerate the sulfidation of mZVI by S0. The S0 with S/Fe molar ratio of 0.1 was fully reacted with mZVI in all solutions to form unevenly distributed FeS species on S-mZVIs as confirmed by SEM-EDX and XANES characterization. The cations depassivated the mZVI surface by driving the proton release from the surface site (FeOH) and resulting in localized acidification. The probe reaction test (tetrachloride dechlorination) and open circuit potential (EOCP) measurement demonstrated that Mg2+ was most efficient in depassivating the mZVI and therefore promoting sulfidation. The decrease of surface proton for hydrogenolysis on the S-mZVI synthesized in MgCl2 solution also inhibited the formation of cis-1,2-dichloroethylene by 14–79% compared to other S-mZVIs during trichloroethylene dechlorination. In addition, the synthesized S-mZVIs exhibited the highest reduction capacity reported so far. These findings provide a theoretical basis for the facile on-site sulfidation of mZVI by S0 with cation-rich natural waters for sustainable remediation of contaminated sites.

Evolutionary diversity of proton and water channels on the oxidizing side of photosystem II and their relevance to function.

One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn4CaO5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the 'water wheel' to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn4CaO5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S0 → S1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421-425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831-15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).

The Ancestral Shape of the Access Proton Path of Mitochondrial ATP Synthases Revealed by a Split Subunit-a.

The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli, resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

Iron toxicity downregulates root-proton efflux and decreases zinc accumulation in cassava

Iron (Fe) plays a pivotal role in biogeochemical cycling in the soil-plant-human continuum; however, much research has focused on Fe deficiency with limited knowledge about Fe toxicity impacts on micronutrient (e.g., zinc: Zn) accumulation in cassava, an important staple tuber food in the Tropics. A sand-hydroponic-hybrid technique was undertaken for 60 days to investigate the effects of variable Fe concentrations (0.1–500 μmol Fe L−1) on the root-proton and root-organic acid release, rhizosphere Fe solubility, and levels of cassava nutrient accumulation. The results showed that high Fe concentrations (50–500 μmol Fe L−1) significantly decreased the root (0.34–0.51 g dry weight) biomass compared to the control (0.82–0.83 g dry weight). The Fe concentrations significantly regulated the net proton release from the roots by upregulating (25–45 μmol L−1 h−1 g−1 fresh weight root) and downregulating (1.8–7.1 μmol L−1 h−1 g−1 fresh weight root) the net proton efflux at low (1.0 μmol Fe L−1) and high Fe concentrations (500 μmol Fe L−1), respectively. Succinic acid was the sole organic acid observed and only in the Fe-sensitive cultivar. The water-extractable Fe in the rhizosphere sand (average 3.37 mg kg−1) was lower than in the bulk sand (average 5.31 mg kg−1), indicating that net proton efflux controlled rhizosphere Fe solubility. The high Fe concentrations significantly decreased Zn accumulation (20–29 mg Zn kg−1) but increased phosphorus (P) accumulation (2.7–3.4 g P kg−1) in the roots compared to the control (48–59 mg Zn kg−1 and 2.0–2.3 g P kg−1, respectively), implying that Fe toxicity could be responsible for the undernourished Zn content in the tuber. This study suggested the importance of alleviating Fe toxicity in soil-plant systems to mitigate cassava root Zn deficiency, which is relevant to human Zn malnutrition in countries consuming cassava as the main staple diet.

Structural evidence for intermediates during O2 formation in photosystem II

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry1–3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S3→[S4]→S0 transition where O2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2→S3 transition4–6, disappears or relocates in parallel with Yz reduction starting at approximately 700 μs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Energetics and proton release in photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA2 or psbA3 gene

In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of PheD1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative Em of PheD1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2-PSII by measuring the thermoluminescence from the S2QA−/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the Em of PheD1–/PheD1 was decreased by ∼30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S2TyrZ• to S3TyrZ and S3TyrZ• → (S3TyrZ•)’ transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.

Biocathode electrochemical activity and reduction characteristics of Cr(VI) system in microbial fuel cells

AbstractBACKGROUNDThe biocathode of microbial fuel cells (MFCs) offers a simple method for the reductive treatment of Cr(VI), and Cr(VI) is an electron acceptor in cathodes in MFCs. In this study, MFCs with different Cr(VI) contents (7.07–35.35 mg L−1) were considered to evaluate the overall performance of MFCs.RESULTSThe results of electrochemical analysis confirmed that the MFC‐2 biocathode exhibited a higher effective reaction surface, stronger conductivity, and smaller charge transfer resistance (53.63 Ω) than the others. In addition, Cr(VI) had a power output of 75.6 mW m−2 in MFC‐2 and a Cr(VI) cathodic coulombic efficiency of 103.8%. With 14.14 mg L−1 Cr(VI) content, the cathode microbe had the best activity. Cr(VI) reduction followed a pseudo‐first‐order kinetic model, with the rate constant being 0.0139 h−1. Meanwhile, this study analyzed the Cr(VI) reduction in MFCs and the release and acceptance of electrons and protons. Electrically active bacteria such as Proteobacteria were highly enriched on the anode and cathode biofilm, while the Cr(VI)‐reducing bacteria Gammaproteobacteria increased in the biofilm of the cathode.CONCLUSIONThis study helps in the selection of Cr(VI) concentration for the cathode, guaranteeing microbial activity and power generation performance. Microbes have the best electrochemical activity in MFC‐2 (14.14 mg L−1). Meanwhile, the change in anode microbial activity was affected by cathode performance. The microbial community was significantly influenced by Cr(VI). © 2023 Society of Chemical Industry (SCI).

Improved rock phosphate dissolution from organic acids is driven by nitrate assimilation of bacteria isolated from nitrate and CaCO3-rich soil.

Until now, the solubilization capacities of insoluble mineral P by soil microorganisms have been screened in vitro with media containing NH4+ as a nitrogen source. This presence of NH4+ will lead to an acidification of the medium responsible for the solubilization of the insoluble P. However, besides proton release, the production of organic acids can play a very important role in the release of free P. This physiological mechanism can largely depend on the source of nitrogen (NH4+vs NO3-) assimilated by the bacteria but the influence of the N source on the production of organic acids has yet to be studied. Our aim was to investigate if the N source assimilated by bacteria and the soil characteristics such as the dominant N source (NH4+vs NO3-) and CaCO3 contents might influence the bacterial capacities to solubilize rock phosphate. To fill this objective, we screened the capacity of bacteria isolated from 3 soils to solubilize rock phosphate in vitro in presence of NH4+or NO3-. Then, we selected the most efficient bacterial strains to identify and quantify the release of organic anions into the medium. Among the two hundred and forty-three bacterial strains isolated from the 3 soils, nine and seven isolates were identified with the highest % rock phosphate-solubilization values with NH4+ or NO3- as the sole N-source. Only one strain was able to release free Pi with NH4+ or NO3- as the sole N-source. The most predominant organic acids released by almost all isolates were gluconic acid, lactic acid, glycolic acid, acetic acid, formic acid and pyruvic acid regardless the N-source. However, with NO3- as source of N, the highest concentrations on those acids were found together with the highest release of free Pi into the medium. Molecular analysis of 16S rRNA indicated that almost all strains belonged to Bacillus and Paenibacillus genera. The PCA analysis between soil properties and bacterial capacities to release organic acids and free Pi also revealed that soil factors such as CaCO3 and soil NO3- content positively influenced the release of organic acids by bacteria grown in vitro. Our results concluded that the bacterial rock phosphate-solubilization was intimately related to organic acids production which in turn seemed to be driven by the assimilation of NO3- by bacteria. Therefore, the N-source might be considered a key factor to take into consideration during the screening and selection of suitable strains involved in the P-solubilization.

Photoacidity of Indolinospirobenzopyrans in Water

AbstractThe reversible isomerism of indolinospirobenzopyrans is perhaps among the most studied phenomena in the field of molecular switches. Although they began to gain attention as early as 70 years ago following the seminal work of Hirshberg and Fischer, who were the first to recognize their photochromic behaviours, their implementation as photoacids emerged prominently only in the last decade. In this Review, we contextualize the prerequisites underlying the photo‐triggered proton release that occurs in these molecular switches, highlighting the most recent advances in their characterization and application as “metastable‐state photoacids” in water.

Comparative Thermodynamics of the Reversible Self-Association of Therapeutic mAbs Reveal Opposing Roles for Linked Proton- and Ion-Binding Events.

Reversible self-association (RSA) has long been a concern in therapeutic monoclonal antibody (mAb) development. Because RSA typically occurs at high mAb concentrations, accurate assessment of the underlying interaction parameters requires explicitly addressing hydrodynamic and thermodynamic nonideality. We previously examined the thermodynamics of RSA for two mAbs, C and E, in phosphate buffered saline (PBS). Here we continue to explore the mechanistic aspects of RSA by examining the thermodynamics of both mAbs under reduced pH and salt conditions. Dynamic light scattering and sedimentation velocity (SV) studies were conducted for both mAbs at multiple protein concentrations and temperatures, with the SV data analyzed via global fitting to determine best-fit models, interaction energetics, and nonideality contributions. We find that mAb C self-associates isodesmically irrespective of temperature, and that association is enthalpically driven but entropically penalized. Conversely, mAb E self-associates cooperatively and via a monomer-dimer-tetramer-hexamer reaction pathway. Moreover, all mAb E reactions are entropically driven and enthalpically modest or minimal. The thermodynamics for mAb C self-association are classically seen as originating from van der Waals interactions and hydrogen bonding. However, relative to the energetics we determined in PBS, self-association must also be linked to proton release and/or ion uptake events. For mAb E, the thermodynamics implicate electrostatic interactions. Furthermore, self-association is instead linked to proton uptake and/or ion release, and primarily by tetramers and hexamers. Finally, although the origins of mAb E cooperativity remain unclear, ring formation remains a possibility whereas linear polymerization reactions can be eliminated.

The Influence of Deep-Level Defects With Various Depths in SiO2 on Ionizing Damage

In this article, we analyzed the interaction mechanism between ionization and displacement defects in SiO2 using experimental and simulation techniques. In the experiment, the gate-controlled lateral p-n-p transistors (GLPNPs) were pretreated by He ions irradiation with different energies, which can create additional oxygen vacancy defects (Vo) at various depth ranges in SiO2. The pretreated GLPNPs were irradiated with <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\gamma $ </tex-math></inline-formula> -rays to analyze their response to ionization damage. It can be found that the introduction of additional deep Vo in SiO2 weakens the ionization damage of GLPNPs, and this weakening effect is heavily related to the depth of additional deep Vo. In addition, the TCAD simulation tool was used to investigate the mechanism by which the ionization damage is weakened with additional deep Vo depth. The simulation results show that as the additional deep Vo defect approaches the SiO2–Si interface, the additional deep Vo defect enhances the capture of the holes (or release of electrons), resulting in a decrease in hydrogenated Vo ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mathbf {Vo}\mathbf {H}_{\mathbf {2}}^{\mathbf {+}}$ </tex-math></inline-formula> ) concentration, which affects the release of protons and ultimately reduces <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$N_{\mathrm {it}}$ </tex-math></inline-formula> . Based on the above mechanism, it would be helpful to accurately evaluate the electrical characteristics degradation of bipolar transistors in complex space environments.

Solar Energetic Particle Events with Short and Long Onset Times

Gradual solar energetic particle (SEP) events, usually attributed to shock waves driven by coronal mass ejections (CMEs), show a wide variety of temporal behaviors. For example, TO, the >10 MeV proton onset time with respect to the launch of the CME, has a distribution of at least an order of magnitude, even when the source region is not far from the so-called well-connected longitudes. It is important to understand what controls TO, especially in the context of space weather prediction. Here we study two SEP events from the western hemisphere that are different in TO on the basis of >10 MeV proton data from the Geostationary Operations Environmental Satellite, despite being similar in the CME speed and longitude of the source regions. We try to find the reasons for different TO, or proton release times, in how the CME-driven shock develops and the Alfvén Mach number of the shock wave reaches some threshold by combining the CME height-time profiles with radio dynamic spectra. We also discuss how CME–CME interactions and active region properties may affect the proton release times.

Ultrathin Ultralow-Platinum Catalyst Coated Membrane for Proton Exchange Membrane Fuel Cells.

Catalyst coated membrane (CCM) is the core component of proton exchange membrane fuel cells and is routinely fabricated by spraying Pt/C slurriesonto membrane, resulting in low activity and thick catalyst layer (CL, 5-10µm) with an unaffordable Pt loading of 0.2-0.4mg cm-2 and a large mass transfer resistance at cathode. Highly active ultrathin ultralow-Pt CL (UUCL) is urgently required, but remains rare. Herein, wet-chemical direct growth of UUCLs on both sides of membrane to achieve integrated ultrathin ultralow-Pt catalyst coated membranes (UUCCMs) with a cathodic CL thickness of 79.7 ± 15.0nm and a Pt loading of 20.2 ± 1.6µg cm-2 is reported. The key to this unique fabrication is the release of proton from membrane to regioselectively initiate the growth of interconnected Pd nanoneedle clusters array on membrane, followed by high-density deposition of Pt nanoparticles on Pd (Pt/Pd UUCLs). The single cell of UUCCMs exhibits the highest mass peak power density of 59.9W mgPt,Cathode -1 in the literature. The exceptional activity originates from high electrochemically active surface area, remarkable oxygen reduction reaction activity closely correlated with strain, and electronic effect at Pt/Pd interface, as well as improved mass transfer and optimal water management.

Highly Robust Nanogels from Thermal-Responsive Nanoparticles with Controlled Swelling for Engineering Deployments

Regular nanogels have been demonstrated their inefficiency for subterranean oil recovery due to their intrinsic drawbacks of fast swelling within minutes, thermal instability, and salinity vulnerability. Prior deployment of swelling delayed nanogels mainly depended on the reservoirs at a relatively higher temperature. To address the issues encountered during engineering deployment, hereinwe devised an integrative approach to in situ form swelling delayed robust nanogels by introducing radically active monomers with thermally sensitive moieties. The nanoparticles with hydrophobic cores in brine in response to thermal input in situ generated well-dispersed hydrophilic nanogels, which showed a pronounced delayed swelling of a week compared to traditional nanogels showing swelling kinetics within minutes. Furthermore, the formation of swelling-delayed nanogels could occur at ambient temperature. This behavior was radically different from that of temperature-controlled labile cross-linkers containing nanogels, requiring temperatures greater than 50 °C for volume increase thanks to ester hydrolysis. In addition, the in-situ formed nanogels displayed long-term thermal stability and salinity tolerance under hostile media at temperatures up to 130 °C. The release of an acidic proton under aqueous conditions has been demonstrated to control the microenvironment for various scenarios. The nanotechnology of converting hydrophobic nanoparticles to hydrophilic nanogels could be applied in a wide range of practical applications such as plugging materials and foaming stabilizers for in-depth conformance control during water and CO2 flooding.

Coumarinacyl Anilinium Salt: A Versatile Visible and NIR Photoinitiator for Cationic and Step-Growth Polymerizations

A coumarinacyl anilinium (CAA) salt, facilely synthesized via a one-pot reaction, is shown to be a versatile visible and NIR photoinitiator for cationic and step-growth polymerizations. CAA salt exhibits superior photoinitiation performance as compared to commercial iodonium salt in cationic polymerization. Upon visible-light irradiation, this salt undergoes hemolytic and heterolytic cleavage and subsequent electron transfer and hydrogen abstraction reactions, forming reactive species capable of initiating cationic polymerization of epoxides and vinyl monomers. After a short irradiation period, polymerization also proceeds in the dark due to the non-nucleophilic nature of the counteranion. NIR-induced polymerizations were successfully conducted based on upconversion photochemistry. CAA salt can also initiate step-growth polymerization of N-ethyl carbazole (NEC) by oxidation of the monomer by the photochemically formed anilium radical cations. Subsequent proton release and radical coupling reactions essentially yield polycarbazole. CAA salt, featuring straightforward synthesis and long-wavelength sensitivity as well as versatile photoinitiating performance, has great potential in various applications.

How does sequence influence charge regulation of disordered proteins?

Charge regulation, defined as the regulation of charge states of ionizable residues through the uptake or release of protons, can influence the conformational ensembles and distributions of charge states that are accessible to intrinsically disordered proteins. These influences are exerted through sequence- and conformational-context dependent pKa values. We recently developed and deployed a q-canonical ensemble that allows for the linked analyses of separate measurements, of charge and conformation as a function of pH. When combined with atomistic, q-canonical Monte Carlo simulations based on the ABSINTH implicit solvation model, we were able to uncover the pH-dependent interplay of charge state and conformational heterogeneity. However, the combinatorial increase in the number of charge states to consider has remained a persistent challenge. Here, we introduce a breakthrough that allows us to predict the extent of charge regulation to be expected for IDPs arbitrary numbers of ionizable residues. The algorithm leverages prior information regarding the effects of local sequence contexts on charge regulation. Longer sequences with arbitrary numbers of ionizable residues are built up using a chain-growth Monte Carlo procedure, each step building on extant information for peptide fragments. We have deployed the new methodology in a high-throughput investigation of sequences drawn from the IDRome, which is the sub-proteome of intrinsically disordered regions (IDRs). We find that the apparent charge, obtained by assuming model compound pKa values, is rarely the same as the real charge that one predicts based on an accounting of charge regulation effects. This work paves the way to understanding the sequence features that result in charge regulation effects, and their importance to biomolecular processes.

Portable All-in-One Electrochemical Actuator-Sensor System for the Detection of Dissolved Inorganic Phosphorus in Seawater.

We present a methodology for the detection of dissolved inorganic phosphorous (DIP) in seawater using an electrochemically driven actuator-sensor system. The motivation for this work stems from the lack of tangible solutions for the in situ monitoring of nutrients in water systems. It does not require the addition of any reagents to the sample and works under mild polarization conditions, with the sample confined to a thin-layer compartment. Subsequent steps include the oxidation of polyaniline to lower the pH, the delivery of molybdate via a molybdenum electrode, and the formation of an electroactive phosphomolybdate complex from DIP species. The phosphomolybdate complex is ultimately detected by either cyclic voltammetry (CV) or square wave voltammetry (SWV). The combined release of protons and molybdate consistently results in a sample pH < 2 as well as a sufficient excess of molybdate, fulfilling the conditions required for the stoichiometric detection of DIP. The current of the voltammetric peak was found to be linearly related to DIP concentrations between 1 and 20 μM for CV and 0.1 and 20 μM for SWV, while also being selective against common silicate interference. The analytical application of the system was demonstrated by the validated characterization of five seawater samples, revealing an acceptable degree of difference compared to chromatography measurements. This work paves the way for the future DIP digitalization in environmental waters by in situ electrochemical probes with unprecedented spatial and temporal resolution. It is expected to provide real-time data on anthropogenic nutrient discharges as well as the improved monitoring of seawater restoration actions.

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.

Membrane composition may regulate proton wave propagation.

Lateral proton diffusion along membrane surfaces, specifically between a proton source and a sink, is a fundamental process in many biological systems. It may play a significant part in the uptake of cations, sugars, amino acids, and ATP synthesis. Fast interfacial diffusion cannot rely on titratable membrane residues, as a comparison with the relatively slow proton release reaction shows [1]. Yet, an entropy-dominated free energy barrier prevents surface protons from equilibrating with bulk protons [2]. The dependence of barrier height on membrane compositions thus far remained enigmatic. To establish the barrier-modulating role of glycolipids, we released protons to a small spot on the surface of planar lipid bilayers by Ca2+ microinjection. The membranes contained a calcium ionophore (A23187) which facilitated the transmembrane transport of Ca2+ ions in exchange for protons [3]. A lipid-anchored pH-sensor allowed detection of the arrival of the locally triggered proton wave on a distant membrane patch as a change in fluorescence intensity. Our results indicated that interfacial sugar moieties might be potent regulators of proton migration between membrane-embedded proton sources and sinks. This project was supported by the Marie Skłodowska-Curie grant 860592 (Horizon 2020). [1] Springer, et al. Proc. Natl. Acad. Sci. U.S.A, 2011, 108, 14461-14466. [2] Weichselbaum, et al. Scientific Reports, 2017, 7, 4553. [3] Peter Pohl, Yuri N. Antonenko, Lev S. Yaguzhinsky, Biochimica et Biophysica Acta, 1990, 1027, 295-300.

An Adapted Protocol for Quantitative Rhizosphere Acidification Assay.

Acidification of the rhizosphere is a key process in the homeostasis of multiple essential nutrients, including iron. Under iron deficiency, the release of protons from the roots helps solubilize and increase the accessibility of iron in the soil. Rhizosphere acidification has been widely examined in many iron homeostasis studies, generally using a qualitative method based on the color change of bromocresol purple, a pH indicator dye, near the roots. In this chapter, we introduce an adapted version of a rhizosphere acidification assay protocol that allows for the quantitative assessment of small pH changes in the rhizosphere. This colorimetric method also utilizes bromocresol purple, but the ratio of its absorbance at 434nm and 588nm is considered to quantify protons released into the assay solution. Furthermore, the assay is compatible with small sample volumes, such as those with young Arabidopsis seedlings.