Structure of Mammalian Respiratory Supercomplex I1III2IV1.

The mammalian respiratory chain complexes assemble into supercomplexes (SCs) and reside in the inner mitochondrial membrane to transfer electrons and establish the proton gradient for complex V to synthesize ATP. The precise arrangement of SCs is largely unknown. Here, we report a 4.0-Å cryo-electron microscopy (cryo-EM) structure of the major SC in porcine heart, the 1.7-MDa SCI1III2IV1. The complex III (CIII) dimer and complex IV (CIV) bind at the same side of the L-shaped complex I (CI). Several accessory or supernumerary subunits of CI, such as NDUFA11, NDUFB4, NDUFB8, and NDUFB9, directly contribute to the oligomerization of CI, CIII, and CIV. COX7C and COX7A of CIV attach CIV to the concave surface formed by CIII and the distal end of membrane arm of CI. The structure suggests a possible mechanism by which electrons are transferred from NADH to cytochrome c and provides a platform for future functional dissection of respiration.

Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

A bacterium from a mountain lake harvests light using both proton-pumping xanthorhodopsins and bacteriochlorophyll-based photosystems.

Photoheterotrophic bacteria harvest light energy using either proton-pumping rhodopsins or bacteriochlorophyll (BChl)-based photosystems. The bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for both systems. Here, we show that BChl is expressed between 4°C and 22°C in the dark, whereas xanthorhodopsin is expressed only at temperatures below 16°C and in the presence of light. Thus, cells grown at low temperatures under a natural light-dark cycle contain both BChl-based photosystems and xanthorhodopsins with a nostoxanthin antenna. Flash photolysis measurements proved that both systems are photochemically active. The captured light energy is used for ATP synthesis and stimulates growth. Thus, S. glacialis AAP5 represents a chlorophototrophic and a retinalophototrophic organism. Our analyses suggest that simple xanthorhodopsin may be preferred by the cells under higher light and low temperatures, whereas larger BChl-based photosystems may perform better at lower light intensities. This indicates that the use of two systems for light harvesting may represent an evolutionary adaptation to the specific environmental conditions found in alpine lakes and other analogous ecosystems, allowing bacteria to alternate their light-harvesting machinery in response to large seasonal changes of irradiance and temperature.

Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Crystallographic cyanide-probing for cytochrome c oxidase reveals structural bases suggesting that a putative proton transfer H-pathway pumps protons.

Cytochrome c oxidase (CcO) reduces O2 in the O2-reduction site by sequential four-electron donations through the low-potential metal sites (CuA and Fea). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp51, Arg438, Glu198, the hydroxyfarnesyl ethyl group of heme a, and Ser382, respectively. These sites interact with the putative proton-pumping H-pathway. However, the metal sites responsible for each structural change have not been identified, since these changes were detected as structural differences between the fully reduced and fully oxidized CcOs. Thus, the roles of these structural changes in the CcO function are yet to be revealed. X-ray crystal structures of cyanide-bound CcOs under various oxidation states showed that the O2-reduction site controlled only the Ser382-including site, while the low-potential metal sites induced the other changes. This finding indicates that these low-potential site-inducible structural changes are triggered by sequential electron-extraction from the low-potential sites by the O2-reduction site and that each structural change is insensitive to the oxidation and ligand-binding states of the O2-reduction site. Because the proton/electron coupling efficiency is constant (1:1), regardless of the reaction progress in the O2-reduction site, the structural changes induced by the low-potential sites are assignable to those critically involved in the proton pumping, suggesting that the H-pathway, facilitating these low-potential site-inducible structural changes, pumps protons. Furthermore, a cyanide-bound CcO structure suggests that a hypoxia-inducible activator, Higd1a, activates the O2-reduction site without influencing the electron transfer mechanism through the low-potential sites, kinetically confirming that the low-potential sites facilitate proton pump.

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping.

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the ß-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site.

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Mitochondria at the Nanoscale: Physics Meets Biology-What Does It Mean for Medicine?

Mitochondria are commonly perceived as "cellular power plants". Intriguingly, power conversion is not their only function. In the first part of this paper, we review the role of mitochondria in the evolution of eukaryotic organisms and in the regulation of the human body, specifically focusing on cancer and autism in relation to mitochondrial dysfunction. In the second part, we overview our previous works, revealing the physical principles of operation for proton-pumping complexes in the inner mitochondrial membrane. Our proposed simple models reveal the physical mechanisms of energy exchange. They can be further expanded to answer open questions about mitochondrial functions and the medical treatment of diseases associated with mitochondrial disorders.

Correlated particle transport enables biological free energy transduction.

Studies of biological transport frequently neglect the explicit statistical correlations among particle site occupancies (i.e., they use a mean-field approximation). Neglecting correlations sometimes captures biological function, even for out-of-equilibrium and interacting systems. We show that neglecting correlations fails to describe free energy transduction, mistakenly predicting an abundance of slippage and energy dissipation, even for networks that are near reversible and lack interactions among particle sites. Interestingly, linear charge transport chains are well described without including correlations, even for networks that are driven and include site-site interactions typical of biological electron transfer chains. We examine three specific bioenergetic networks: a linear electron transfer chain (as found in bacterial nanowires), a near-reversible electron bifurcation network (as in complex III of respiration and other recently discovered structures), and a redox-coupled proton pump (as in complex IV of respiration).

Targeting ATP12A, a Nongastric Proton Pump α Subunit, for Idiopathic Pulmonary Fibrosis Treatment.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition of unknown etiology that results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear, and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to cystic fibrosis lung disease. The ATP12A gene encodes the α-subunit of the nongastric H+, K+-ATPase, which functions to acidify the airway surface fluid and impairs mucociliary transport function in patients with cystic fibrosis. It is hypothesized that the ATP12A protein may play a role in the pathogenesis of IPF. The authors' studies demonstrate that ATP12A protein is overexpressed in distal small airways from the lungs of patients with IPF compared with normal human lungs. In addition, overexpression of the ATP12A protein in mouse lungs worsened bleomycin induced experimental pulmonary fibrosis. This was prevented by a potassium competitive proton pump blocker, vonoprazan. These data support the concept that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has potential as a novel therapeutic strategy in IPF treatment.

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography.

DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.

Nuclear Magnetic Resonance Detection of Hydrogen Bond Network in a Proton Pump Rhodopsin RxR and Its Alteration during the Cyclic Photoreaction.

Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.

Converting a Natural-Light-Driven Outward Proton Pump Rhodopsin into an Artificial Inward Proton Pump.

Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H+) pumps, new subfamilies of microbial rhodopsins transporting H+ inwardly, i.e., light-driven inward H+ pumps, have been discovered recently. Although structural and spectroscopic studies provide insights into their ion transport mechanisms, the minimum key element(s) that determine the direction of H+ transport have not yet been clarified. Here, we conducted the first functional conversion study by substituting key amino acids in a natural outward H+-pumping rhodopsin (PspR) with those in inward H+-pumping rhodopsins. Consequently, an artificial inward H+ pump was constructed by mutating only three residues of PspR. This result indicates that these residues govern the key processes that discriminate between outward and inward H+ pumps. Spectroscopic studies revealed the presence of an inward H+-accepting residue in the H+ transport pathway and direct H+ uptake from the extracellular solvent. This finding of the simple element for determining H+ transport would provide a new basis for understanding the concept of ion transport not only by microbial rhodopsins but also by other ion-pumping proteins.

Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I.

Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 µs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes.

Opioid Use in Murine Model Results in Severe Gastric Pathology that May Be Attenuated by Proton Pump Inhibition.

Opioids are the gold standard for chronic and acute pain management; however, their consequence on gastric function is relatively understudied. Opioid users have a higher incidence of gastric dysfunction, worse quality of life, increased hospitalizations, and increased use of antiemetic and pain modulator medications. The current study shows that morphine treatment in the murine model results in greater disruption of gastric epithelial cell morphology, increased gastric cell apoptosis, elevated inflammatory cytokines, and matrix metallopeptidase-9 secretion. Morphine treatment also increases gastric acid secretion and causes delays in gastric emptying. Moreover, morphine treatment causes an increase in systemic IL-6 level, which plays an important role in morphine-induced delayed gastric emptying and gastric damage. IL-6 knockout mice show a significant level of reduction in morphine-induced gastric delaying, acid retention, and gastric damage. Thus, morphine-mediated gastric damage is a consequence of the accumulation of acid in the stomach due to increased gastric acid secretion and delayed gastric emptying. Treatment with a proton pump inhibitor resulted in a significant reduction in morphine-induced gastric inflammation, gastric delaying, and improved morphine tolerance. Hence, these studies attribute morphine-mediated induction in gastric acidity and inflammatory cytokines as drivers for morphine-associated gastric pathology and show the therapeutic use of proton pump inhibitors as an inexpensive approach for clinical management of morphine-associated pathophysiology and analgesic tolerance.

Receptor-like protein kinase BAK1 promotes K+ uptake by regulating H+-ATPase AHA2 under low potassium stress.

Potassium (K+) is one of the essential macronutrients for plant growth and development. However, the available K+ concentration in soil is relatively low. Plant roots can perceive low K+ (LK) stress, then enhance high-affinity K+ uptake by activating H+-ATPases in root cells, but the mechanisms are still unclear. Here, we identified the receptor-like protein kinase Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) that is involved in LK response by regulating the Arabidopsis (Arabidopsis thaliana) plasma membrane H+-ATPase isoform 2 (AHA2). The bak1 mutant showed leaf chlorosis phenotype and reduced K+ content under LK conditions, which was due to the decline of K+ uptake capacity. BAK1 could directly interact with the AHA2 C terminus and phosphorylate T858 and T881, by which the H+ pump activity of AHA2 was enhanced. The bak1 aha2 double mutant also displayed a leaf chlorosis phenotype that was similar to their single mutants. The constitutively activated form AHA2Δ98 and phosphorylation-mimic form AHA2T858D or AHA2T881D could complement the LK sensitive phenotypes of both aha2 and bak1 mutants. Together, our data demonstrate that BAK1 phosphorylates AHA2 and enhances its activity, which subsequently promotes K+ uptake under LK conditions.

New insights into the proton pumping mechanism of ba3 cytochrome c oxidase: the functions of key residues and water.

As the terminal oxidase of cell respiration in mitochondria and aerobic bacteria, the proton pumping mechanism of ba3-type cytochrome c oxidase (CcO) of Thermus thermophiles is still not fully understood. Especially, the functions of key residues which were considered as the possible proton loading sites (PLSs) above the catalytic center, as well as water located above and within the catalytic center, remain unclear. In this work, molecular dynamic simulations were performed on a set of designed mutants of key residues (Asp287, Asp372, His376, and Glu126II). The results showed that Asp287 may not be a PLS, but it could modulate the ability of the proton transfer pathway to transfer protons through its salt bridge with Arg225. Maintaining the closed state of the water pool above the catalytic center is necessary for the participation of inside water molecules in proton transfer. Water molecules inside the water pool can form hydrogen bond chains with PLS to facilitate proton transfer. Additional quantum cluster models of the Fe-Cu metal catalytic center are established, indicating that when the proton is transferred from Tyr237, it is more likely to reach the OCu atom directly through only one water molecule. This work provides a more profound understanding of the functions of important residues and specific water molecules in the proton pumping mechanism of CcO.

F-type proton-pumping ATPase mediates acid tolerance in Streptococcus mutans.

AIMS: Streptococcus mutans is highly sensitive to inhibitors of proton-pumping F-type ATPase (F-ATPase) under acidic conditions. Herein, we investigated the role of S. mutans F-ATPase in acid tolerance using a bacterium expressing the F-ATPase ß subunit at lower levels than the wild-type strain. METHODS AND RESULTS: We generated a mutant S. mutans expressing the catalytic ß subunit of F-ATPase at lower levels than the wild-type bacterium. The mutant cells exhibited a significantly slower growth rate at pH 5.30, whereas the rate was essentially the same as that of wild-type cells at pH 7.40. In addition, the colony-forming ability of the mutant was decreased at pH <4.30 but not at pH 7.40. Thus, the growth rate and survival of S. mutans expressing low levels of the ß subunit were reduced under acidic conditions. CONCLUSIONS: Together with our previous observations, this study indicates that F-ATPase is involved in the acid tolerance mechanism of S. mutans by secreting protons from the cytoplasm.

Features of the Mechanism of Proton Transport in ESR, Retinal Protein from Exiguobacterium sibiricum.

Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.