Electrochemical Proton Gradient Research Articles

Optimization of ATP Synthase c-Rings for Oxygenic Photosynthesis.

The conversion of sunlight into useable cellular energy occurs via the proton–coupled electron transfer reactions of photosynthesis. Light is absorbed by photosynthetic pigments and transferred to photochemical reaction centers to initiate electron and proton transfer reactions to store energy in a redox gradient and an electrochemical proton gradient (proton motive force, pmf), composed of a concentration gradient (ΔpH) and an electric field (Δψ), which drives the synthesis of ATP through the thylakoid FoF1-ATP synthase. Although ATP synthase structure and function are conserved across biological kingdoms, the number of membrane–embedded ion–binding c subunits varies between organisms, ranging from 8 to 17, theoretically altering the H+/ATP ratio for different ATP synthase complexes, with profound implications for the bioenergetic processes of cellular metabolism. Of the known c–ring stoichiometries, photosynthetic c–rings are among the largest identified stoichiometries, and it has been proposed that decreasing the c-stoichiometry could increase the energy conversion efficiency of photosynthesis. Indeed, there is strong evidence that the high H+/ATP of the chloroplast ATP synthase results in a low ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio produced by photosynthetic linear electron flow, requiring secondary processes such as cyclic electron flow to support downstream metabolism. We hypothesize that the larger c subunit stoichiometry observed in photosynthetic ATP synthases was selected for because it allows the thylakoid to maintain pmf in a range where ATP synthesis is supported, but avoids excess Δψ and ΔpH, both of which can lead to production of reactive oxygen species and subsequent photodamage. Numerical kinetic simulations of the energetics of chloroplast photosynthetic reactions with altered c–ring size predicts the energy storage of pmf and its effects on the photochemical reaction centers strongly support this hypothesis, suggesting that, despite the low efficiency and suboptimal ATP/NADPH ratio, a high H+/ATP is favored to avoid photodamage. This has important implications for the evolution and regulation of photosynthesis as well as for synthetic biology efforts to alter photosynthetic efficiency by engineering the ATP synthase.

The Evaluation of Mitochondrial Membrane Potential Using Fluorescent Dyes or a Membrane-Permeable Cation (TPP+) Electrode in Isolated Mitochondria and Intact Cells.

The proton electrochemical gradient generated by respiratory chain activity accounts for over 90% of all available ATP and, as such, its evaluation and accurate measurements regarding its total values and fluctuations is an invaluable component in the understanding of mitochondrial functions. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulations and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. Here we discuss the advantages and disadvantages of several of these methods, ranging from one that is dependent on the movement of a particular ion (tetraphenylphosphonium (TPP+) with a selective electrode) to the selection of a fluorescent dye from various types to achieve the same goal. The evaluation of the accumulation and movements of TPP+ across the inner mitochondrial membrane, or the fluorescence of accumulated dye particles, is a sensitive and accurate method of evaluating the Δψ in respiring mitochondria (either isolated or still inside the cell).

The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).

In silico simulation of reversible and irreversible swelling of mitochondria: The role of membrane rigidity

Mitochondria have been widely accepted as the main source of ATP in the cell. The inner mitochondrial membrane (IMM) is important for the maintenance of ATP production and other functions of mitochondria. The electron transport chain (ETC) generates an electrochemical gradient of protons known as the proton-motive force across the IMM and thus produces the mitochondrial membrane potential that is critical to ATP synthesis. One of the main factors regulating the structural and functional integrity of the IMM is the changes in the matrix volume. Mild (reversible) swelling regulates mitochondrial metabolism and function; however, excessive (irreversible) swelling causes mitochondrial dysfunction and cell death. The central mechanism of mitochondrial swelling includes the opening of non-selective channels known as permeability transition pores (PTPs) in the IMM by high mitochondrial Ca2+ and reactive oxygen species (ROS). The mechanisms of reversible and irreversible mitochondrial swelling and transition between these two states are still unknown. The present study elucidates an upgraded biophysical model of reversible and irreversible mitochondrial swelling dynamics. The model provides a description of the PTP regulation dynamics using an additional differential equation. The rigidity tensor was used in numerical simulations of the mitochondrial parameter dynamics with different initial conditions defined by Ca2+ concentration in the sarco/endoplasmic reticulum. We were able to estimate the values of the IMM rigidity tensor components by fitting the model to the previously reported experimental data. Overall, the model provides a better description of the reversible and irreversible mitochondrial swelling dynamics.

Proton Control of Transitions in an Amino Acid Transporter

Amino acid transport into the cell is often coupled to the proton electrochemical gradient, as found in the solute carrier 36 family of proton-coupled amino acid transporters. Although no structure of a human proton-coupled amino acid transporter exists, the crystal structure of a related homolog from bacteria, GkApcT, has recently been solved in an inward-occluded state and allows an opportunity to examine how protons are coupled to amino acid transport. Our working hypothesis is that release of the amino acid substrate is facilitated by the deprotonation of a key glutamate residue (E115) located at the bottom of the binding pocket, which forms part of the intracellular gate, allowing the protein to transition from an inward-occluded to an inward-open conformation. During unbiased molecular dynamics simulations, we observed a transition from the inward-occluded state captured in the crystal structure to a much more open state, which we consider likely to be representative of the inward-open state associated with substrate release. To explore this and the role of protons in these transitions, we have used umbrella sampling to demonstrate that the transition from inward occluded to inward open is more energetically favorable when E115 is deprotonated. That E115 is likely to be protonated in the inward-occluded state and deprotonated in the inward-open state is further confirmed via the use of absolute binding free energies. Finally, we also show, via the use of absolute binding free energy calculations, that the affinity of the protein for alanine is very similar regardless of either the conformational state or the protonation of E115, presumably reflecting the fact that all the key interactions are deep within the binding cavity. Together, our results give a detailed picture of the role of protons in driving one of the major transitions in this transporter.

The H channel is not a proton transfer path in yeast cytochrome c oxidase

Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.

Physiopathological Role of the Vesicular Nucleotide Transporter (VNUT) in the Central Nervous System: Relevance of the Vesicular Nucleotide Release as a Potential Therapeutic Target.

Vesicular storage of neurotransmitters, which allows their subsequent exocytotic release, is essential for chemical transmission in the central nervous system. Neurotransmitter uptake into secretory vesicles is carried out by vesicular transporters, which use the electrochemical proton gradient generated by a vacuolar H+-ATPase to drive neurotransmitter vesicular accumulation. ATP and other nucleotides are relevant extracellular signaling molecules that participate in a variety of biological processes. Although the active transport of nucleotides into secretory vesicles has been characterized from the pharmacological and biochemical point of view, the protein responsible for such vesicular accumulation remained unidentified for some time. In 2008, the human SLC17A9 gene, the last identified member of the SLC17 transporters, was found to encode the vesicular nucleotide transporter (VNUT). VNUT is expressed in various ATP-secreting cells and is able to transport a wide variety of nucleotides in a vesicular membrane potential-dependent manner. VNUT knockout mice lack vesicular storage and release of ATP, resulting in blockage of the purinergic transmission. This review summarizes the current studies on VNUT and analyzes the physiological relevance of the vesicular nucleotide transport in the central nervous system. The possible role of VNUT in the development of some pathological processes, such as chronic neuropathic pain or glaucoma is also discussed. The putative involvement of VNUT in these pathologies raises the possibility of the use of VNUT inhibitors for therapeutic purposes.

Single-Cell Bacterial Electrophysiology Reveals Mechanisms of Stress-Induced Damage

An electrochemical gradient of protons, or proton motive force (PMF), is at the basis of bacterial energetics. It powers vital cellular processes and defines the physiological state of the cell. Here, we use an electric circuit analogy of an Escherichia coli cell to mathematically describe the relationship between bacterial PMF, electric properties of the cell membrane, and catabolism. We combine the analogy with the use of bacterial flagellar motor as a single-cell “voltmeter” to measure cellular PMF in varied and dynamic external environments (for example, under different stresses). We find that butanol acts as an ionophore and functionally characterize membrane damage caused by the light of shorter wavelengths. Our approach coalesces noninvasive and fast single-cell voltmeter with a well-defined mathematical framework to enable quantitative bacterial electrophysiology.

Comparative Characterization of the Atlantic salmon, Salmo salar L., Di/Tripeptide Transporters PepT1a and PepT1b

Amino acids and small peptides in the form of di/tripeptides are key nutrients in animal diets and essential contributors to growth. In mammals, di/tripeptides represent a highly significant fraction of dietary protein after digestion and are transported across the brush‐border membrane of the intestinal epithelium via a single transport protein, the Peptide Transporter 1 (PepT1), a member of the Solute Carrier 15 (Slc15) family. PepT1 is a low‐affinity/high‐capacity transporter that uses an inwardly‐directed proton electrochemical gradient to drive the uptake of di/tripeptides, and peptidomimetics, across the cell membrane. However, in teleost fishes two pept1 genes, namely pept1b and pept1a, have been identified. PepT1b has been studied in many teleost species, including the Atlantic (A.) salmon (Salmo salar L.), whereas PepT1a remain unexplored. The aim of our study was to characterize, the A. salmon PepT1a vs. PepT1b in terms of tissue/organ distribution and function, and possibly extrapolate their physiological role(s).Despite the extra (4th) whole genome duplication (WGD) in the Actinopterygian lineage, A. salmon has only two pept1 genes (namely pept1a and pept1b), like in other telosts that didn't undergo the extra WGD, e.g., the zebrafish (Danio rerio). At the amino acid level, the two paralogues share 77% similarity, 64% identity. Tissue distribution and relative abundance of A. salmon pept1a and pept1b revealed that both transcripts have a similar expression profile. A. salmon pept1a is highly expressed in the proximal intestine (pyloric caeca ≈ anterior midgut > midgut >> posterior midgut), but ~5‐fold less than pept1b, which is expressed in the same gut regions. No expression signals were detected in the anterior and posterior stomach or the anterior and posterior hindgut.As assessed by heterologous expression in Xenopus laevis oocytes and Two‐Electrode Voltage Clamp measurements, A. salmon PepT1a is, like PepT1b, a low‐affinity/high‐capacity system, able to sustain an electrogenic, sodium‐independent and pH‐dependent transport of di/tripeptides. Transport associated currents show that the protein exhibits a K0.5 value for the reference dipeptide Gly‐Gln of 0.5 mM at pH 7.6 and of 0.07 mM at pH 6.5.The pre‐steady state and steady‐state transport currents analysis indicate important functional differences between A. salmon PepT1a and PepT1b transporters. The difference in expression levels and a significantly dissimilar substrate specificity for certain di/tripeptides, such as lysine‐containing peptides, supports the idea of distinct roles in peptide recognition and transport for PepT1a and PepT1b.Support or Funding InformationFunded by RFFV; 247978 SalmoFeedPlus and CargillThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Understanding the Role of Polar Residues on the c‐Ring of E. coli F1Fo ATP Synthase

F1Fo ATP synthases catalyze the synthesis of adenosine triphosphate (ATP), the universal biological energy carrier, and are thus integral to life. The membrane‐embedded Fo sector utilizes the proton electrochemical gradient to generate rotation in the rotor subunits, which drives ATP synthesis in the F1 sector. In addition to ATP synthesis, F1Fo can also hydrolyze ATP to pump protons to create an electrochemical gradient. Recent structural models of ATP synthase from cryo‐electron microscopy have revealed the architecture of the rotor‐stator interface; however, the intricacies of the rotation mechanism are not fully understood. Previous research has identified specific residues of the c subunit of E. coli F1Fo, including Arg50 and Thr51 on the cytoplasmic end of transmembrane helix 2, that may be necessary for the H+ translocation mechanism. Mutation of Arg50 or Thr51 to Cys abolished ATP‐driven H+ pumping and blocked H+‐permeability. Interestingly, these mutants were able to grow in succinate minimal medium, suggesting that ATP synthesis activity was unaffected. Arg50 is part of a proposed charge interaction between subunits a and c, and the polar Thr51 may also participate in this interaction. Therefore, we studied the importance of positive charge and polarity at positions 50 and 51 using chemical and genetic modifications. Chemically appending positive charge onto Cys50 with methanethiosulfonate did not restore ATP‐driven H+ pumping activity. However, mutation of Arg50 to Lys or His supported activity. Mutation of Thr51 to Ser or Asp also supported H+ pumping activity. These studies provide insight into the role of polar residues on the surface of the c‐ring and their contribution to the H+‐driven rotational mechanism of F1Fo ATP synthase.Support or Funding InformationSupported by the North Carolina GlaxoSmithKline Foundation and the University of North Carolina Asheville Undergraduate Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial matrix pH acidifies during anoxia and is maintained by the F1Fo-ATPase in anoxia-tolerant painted turtle cortical neurons.

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine‐123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF‐1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo‐ATPase was inhibited with oligomycin‐A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

Effect of Ionization State on Voltage-Sensor Structure in Resting State of the Hv1 Channel.

Voltage-gated proton-selective channels (Hv1) mediate proton extrusion during intracellular acidification. Hv1 is gated by the proton electrochemical gradient. Intracellular ionizable residues in Hv1 have been proposed to serve as proton-binding sites for pH-dependent gating, but detailed descriptions remain unclear. Here, molecular dynamics (MD) simulations were performed to investigate the effect of ionization states of charged residues on the X-ray structure of Hv1. Modification of the protonation state of acidic residues affected the resting conformation of Hv1 by disrupting salt bridges between S4 and the other segments. Upon protonation, conformational changes enabled the displacement of the S4 arginines toward the extracellular side and increased the mobility of hydrophobic residues at the gate. The aqueous crevice was considerably wider with increased hydration in the pore. Solvation free energies of the pore residues were low at the extra- and intracellular entrances, whereas the narrowest region exhibited the energy barrier for water translocation. Our MD data showed that water molecules in the upper and lower pore oriented differently. In neutral pH, the pore water oriented its dipole pointing away from the voltage-sensing domain center, whereas the opposite direction of the water dipole was observed in acidic pH.

T-cell mitochondrial dysfunction and lymphopenia in DOCK2-deficient patients

This study delineates the mitochondrial defects in DOCK2-deficient T cells contributing to the T cell lymphopenia characteristic of this primary immunodeficiency.

Co-expression of SpSOS1 and SpAHA1 in transgenic Arabidopsis plants improves salinity tolerance

BackgroundNa+ extrusion from cells is important for plant growth in high saline environments. SOS1 (salt overly sensitive 1), an Na+/H+ antiporter located in the plasma membrane (PM), functions in toxic Na+ extrusion from cells using energy from an electrochemical proton gradient produced by a PM-localized H+-ATPase (AHA). Therefore, SOS1 and AHA are involved in plant adaption to salt stress.ResultsIn this study, the genes encoding SOS1 and AHA from the halophyte Sesuvium portulacastrum (SpSOS1 and SpAHA1, respectively) were introduced together or singly into Arabidopsis plants. The results indicated that either SpSOS1 or SpAHA1 conferred salt tolerance to transgenic plants and, as expected, Arabidopsis plants expressing both SpSOS1 and SpAHA1 grew better under salt stress than plants expressing only SpSOS1 or SpAHA1. In response to NaCl treatment, Na+ and H+ in the roots of plants transformed with SpSOS1 or SpAHA1 effluxed faster than wild-type (WT) plant roots. Furthermore, roots co-expressing SpSOS1 and SpAHA1 had higher Na+ and H+ efflux rates than single SpSOS1/SpAHA1-expressing transgenic plants, resulting in the former amassing less Na+ than the latter. As seen from comparative analyses of plants exposed to salinity stress, the malondialdehyde (MDA) content was lowest in the co-transgenic SpSOS1 and SpAHA1 plants, but the K+ level was the highest.ConclusionThese results suggest SpSOS1 and SpAHA1 coordinate to alleviate salt toxicity by increasing the efficiency of Na+ extrusion to maintain K+ homeostasis and protect the PM from oxidative damage induced by salt stress.

Slow Ca2+ Efflux by Ca2+/H+ Exchange in Cardiac Mitochondria Is Modulated by Ca2+ Re-uptake via MCU, Extra-Mitochondrial pH, and H+ Pumping by FOF1-ATPase.

Mitochondrial (m) Ca2+ influx is largely dependent on membrane potential (ΔΨm), whereas mCa2+ efflux occurs primarily via Ca2+ ion exchangers. We probed the kinetics of Ca2+/H+ exchange (CHEm) in guinea pig cardiac muscle mitochondria. We tested if net mCa2+ flux is altered during a matrix inward H+ leak that is dependent on matrix H+ pumping by ATPm hydrolysis at complex V (FOF1-ATPase). We measured [Ca2+]m, extra-mitochondrial (e) [Ca2+]e, ΔΨm, pHm, pHe, NADH, respiration, ADP/ATP ratios, and total [ATP]m in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca2+ after adding DNP and CaCl2 is dependent on whether the ΔpHm gradient is/is not maintained by reciprocal outward H+ pumping by complex V. We found that adding CaCl2 enhanced DNP-induced increases in respiration and decreases in ΔΨm while [ATP]m decreased, ΔpHm gradient was maintained, and [Ca2+]m continued to increase slowly, indicating net mCa2+ influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl2 caused larger declines in ΔΨm as well as a slow fall in pHm to near pHe while [Ca2+]m continued to decrease slowly, indicating net mCa2+ efflux in exchange for H+ influx (CHEm) until the ΔpHm gradient was abolished. The kinetics of slow mCa2+ efflux with slow H+ influx via CHEm was also observed at pHe 6.9 vs. 7.6 by the slow fall in pHm until ΔpHm was abolished; if Ca2+ reuptake via the MCU was also blocked, mCa2+ efflux via CHEm became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHEm activity is driven largely by the ΔpHm chemical gradient with H+ leak, while mCa2+ entry via MCU depends largely on the charge gradient ΔΨm. A fall in ΔΨm with excess mCa2+ loading can occur during cardiac cell stress. Cardiac cell injury due to mCa2+ overload may be reduced by temporarily inhibiting FOF1-ATPase from pumping H+ due to ΔΨm depolarization. This action would prevent additional slow mCa2+ loading via MCU and permit activation of CHEm to mediate efflux of mCa2+.HIGHLIGHTS -We examined how slow mitochondrial (m) Ca2+ efflux via Ca2+/H+ exchange (CHEm) is triggered by matrix acidity after a rapid increase in [Ca2+]m by adding CaCl2 in the presence of dinitrophenol (DNP) to permit H+ influx, and oligomycin (OMN) to block H+ pumping via FOF1-ATP synthase/ase (complex V).-Declines in ΔΨm and pHm after DNP and added CaCl2 were larger when complex V was blocked.-[Ca2+]m slowly increased despite a fall in ΔΨm but maintained pHm when H+ pumping by complex V was permitted.-[Ca2+]m slowly decreased and external [Ca2+]e increased with declines in both ΔΨm and pHm when complex V was blocked.-ATPm hydrolysis supports a falling pHm and redox state and promotes a slow increase in [Ca2+]m.-After rapid Ca2+ influx due to a bolus of CaCl2, slow mCa2+ efflux by CHEm occurs directly if pHe is low.

Electrostatic Lock in the Transport Cycle of the Multidrug Resistance Transporter EmrE

EmrE is a small, homodimeric membrane transporter that exploits the established electrochemical proton gradient across the Escherichia coli inner membrane to export toxic polyaromatic cations, prototypical of the wider small-multidrug resistance transporter family. While prior studies have established many fundamental aspects of the specificity and rate of substrate transport in EmrE, low resolution of available structures has hampered identification of the transport coupling mechanism. Here we present a complete, refined atomic structure of EmrE optimized against available cryo-electron microscopy (cryo-EM) data to delineate the critical interactions by which EmrE regulates its conformation during the transport process. With the model, we conduct molecular dynamics simulations of the transporter in explicit membranes to probe EmrE dynamics under different substrate loading and conformational states, representing different intermediates in the transport cycle. The refined model is stable under extended simulation. The water dynamics in simulation indicate that the hydrogen-bonding networks around a pair of solvent-exposed glutamate residues (E14) depend on the loading state of EmrE. One specific hydrogen bond from a tyrosine (Y60) on one monomer to a glutamate (E14) on the opposite monomer is especially critical, as it locks the protein conformation when the glutamate is deprotonated. The hydrogen bond provided by Y60 lowers the [Formula: see text] of one glutamate relative to the other, suggesting both glutamates should be protonated for the hydrogen bond to break and a substrate-free transition to take place. These findings establish the molecular mechanism for the coupling between proton transfer reactions and protein conformation in this proton-coupled secondary transporter.

Design and assembly of a chemically switchable and fluorescently traceable light-driven proton pump system for bionanotechnological applications

Energy-supplying modules are essential building blocks for the assembly of functional multicomponent nanoreactors in synthetic biology. Proteorhodopsin, a light-driven proton pump, is an ideal candidate to provide the required energy in form of an electrochemical proton gradient. Here we present an advanced proteoliposome system equipped with a chemically on-off switchable proteorhodopsin variant. The proton pump was engineered to optimize the specificity and efficiency of chemical deactivation and reactivation. To optically track and characterize the proteoliposome system using fluorescence microscopy and nanoparticle tracking analysis, fluorescenlty labelled lipids were implemented. Fluorescence is a highly valuable feature that enables detection and tracking of nanoreactors in complex media. Cryo-transmission electron microscopy, and correlative atomic force and confocal microscopy revealed that our procedure yields polylamellar proteoliposomes, which exhibit enhanced mechanical stability. The combination of these features makes the presented energizing system a promising foundation for the engineering of complex nanoreactors.

Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis.

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

172 - Novel optogenetic control of mitochondrial energetics rescues electron transport chain inhibition

Mitochondria use an electrochemical proton gradient to produce ATP, the main cellular energy source. This gradient, called the protonmotive force (PMF), also controls substrate uptake, calcium signaling, and reactive oxygen species (ROS) production and signaling. Despite the central role of the PMF in mitochondrial bioenergetics, current tools to modulate the PMF lack spatial and temporal control in vivo. For example, protonophores are used to irreversibly decrease the PMF, but this pharmacologic approach lacks tissue and mitochondria-selectivity. Importantly, there are no means to increase the PMF. Here, we characterize an optogenetic approach to selectively increase the PMF and affect mitochondrial function in vivo with light. Using a novel mitochondria-targeting strategy we expressed a light-activated proton pump in mitochondria of Caenorhabditis elegans. In response to light, the mitochondria-targeted light-activated proton pump (mtLAPP) increased the PMF in isolated mitochondria and whole animals, observed through tetramethylrhodamine ethyl ester fluorescence. Additionally, optogenetic stimulation of bioenergetics by mtLAPP can overcome toxicity from inhibition of proton-pumping complexes of the mitochondrial electron transport chain. Our results provide a novel method to enhance mitochondrial bioenergetics in whole organisms, and may be used to affect physiologic outputs downstream of the PMF, such as ROS signaling and metabolism.

Costs of being hungry in a fast‐paced world

Costs of being hungry in a fast‐paced world