Photosystem II (PSII) catalyzes water oxidation and oxygen evolution by a light-induced electron transfer chain, leading to the generation of electrons, protons and dioxygen. D1-S264 is a residue located close to the QB-binding site, and mutation of this residue has been shown to bring significant effects on the electron transfer and oxygen-evolving activities. Here we analyzed the structure of a Thermosynechococcus elongatus mutant PsbA3-S264V by cryo-electron microscopy at 1.96 Å resolution, which showed significant changes in the structure surrounding the bicarbonate and QB-binding region. Due to change of Ser to Val, the hydrogen-bond between the QB carbonyl oxygen and S264 is altered, which changed the protonation pathway of QB from the original route of D1-H252 through D1-S264 to QB, to a new, longer and less efficient route of D1-H252 through D1-F265 to QB. Two residues, D1-E244 and D2-E242, changed their side chain orientations significantly. Among them, D2-E242 adopted two conformations, and both are largely deviated from the original structure. All these changes led to alterations in hydrogen-bonding networks of two channels, channel A and channel B, that connect the stromal surface to QB and may function to transport protons to protonate QB. Furthermore, isothermal titration calorimetry experiments showed a diminished 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) binding affinity of the mutated PSII, which may be explained by a structural rotation of D1-F255 in the mutant based on structural analysis of DCMU-bound PSII. These findings offer valuable insights into the functions of D1-S264 in QB protonation and function, as well as in the DCMU-binding.
In homeotherms, heat loss and the need for heat production are increased in small animals compared with large ones, and mitochondria are the primary organelles responsible for heat production in animals. Because of this, in animals of different body sizes, mitochondria may differ in their sensitivity to temperature and ability to produce heat via ATP uncoupling. Here, we used primary fibroblast cells isolated from large breed puppies and senior dogs, and small breed puppies and senior dogs to determine whether mitochondria across body sizes and ages in dogs function similarly, or whether smaller breeds tend to be more uncoupled and temperature sensitive due to their whole-animal phenotype of low body temperature and high mass specific metabolic rate. We compared metabolic rates in cells exposed to 37 and 40 °C to determine thermal sensitivities. We found that basal respiration and proton leak increased across all size and age classes in the 40 °C group, while coupling efficiency and spare respiratory capacity decreased, indicating that higher temperatures uncouple mitochondria in dogs, but body size does not affect temperature sensitivity. Additionally, to elucidate mechanistic differences in uncoupling, we treated cells with the UCP2 inhibitor genipin and found that genipin-treated cells increased proton leak in small senior dogs, but reduced proton leak in large puppies. The different responses to UCP2 inhibition correlate with metabolomic differences previously found in dogs of different size and age classes, and our data suggest that the role of UCP2 is dependent on the metabolic phenotype of a cell.
Changes in the level and the ratio of adenine nucleotides (AN) are a common consequence of ischemia, which can promote mitochondrial permeability transition pore (mPTP) opening and cell death upon reperfusion. However, the mechanism of AN-dependent mPTP inhibition is not entirely clear. Here we studied the effects of inorganic phosphate, Mg2+, as well as the inhibitors of adenylate translocase (ANT) and FoF1-ATP synthase (F-ATPase) on the AN-dependent mPTP suppression and AN turnover mediated by a short Ca2+-dependent mitochondrial carrier (SCaMC). Also, we indirectly assessed the contribution of Ca2+ buffering by AN to mPTP suppression. We found that, at near-physiologic concentrations, AN suppressed mPTP opening (swelling) and increased the Ca2+-retention capacity much stronger than the ANT inhibitor bongkrekic acid (BA). Inorganic phosphate (Pi) and Mg2+ modulated the protective effect of AN. In solution, AN were an incomparably weaker Ca2+ buffer than matrix Pi. AN preserved the capability to suppress mPTP opening in the presence of both BA and carboxyatractyloside (CATR). The sensitivity of AN-dependent mPTP suppression to CATR decreased with a decrease in the Pi level. Mg2+ and BA, in contrast to CATR, partially inhibited the SCaMC-mediated AN turnover. The analysis of these and the earlier obtained data allowed us to propose a new mechanism of AN-dependent mPTP suppression: the coordinated ANT- and SCaMC-mediated AN turnover, which fine-tunes the Pi, Ca2+, and H+ ratios in the matrix for efficient Ca2+ sequestration. The mechanism does not require ANT stabilization in any conformation, allosteric regulators, and the formation of AN-Ca2+-Pi complexes.
Respiratory Complex I powers oxidative phosphorylation by a long-range proton-coupled electron transfer (PCET) reaction, with mutations linked to more than half of all human mitochondrial disorders. Yet, the molecular principles underlying the functional impairment remain difficult to test, as most mutations impede both the proton pumping and oxidoreductase activities due to the tightly coupled PCET process. Here, we probe how key disease mutations in the terminal ND5 subunit (NuoL/Nqo12), linked to the development of Leigh's syndrome (LS) and LHON/MELAS (F124L, M252T, D393N), affect the proton transport activity within the dissected antiporter module Nqo12. All constructs result in fully folded antiporter modules, with the introduced substitutions showing enhanced proton conduction rates across the proteoliposome membranes relative to the wild type module. Our molecular dynamics simulations reveal that the mutations perturb the internal water network and ion-pair dynamics that are central for the long-range PCET activity in Complex I. Taken together, we suggest that the mitochondrial disease mutations alter the redox-driven proton pumping activity of Complex I by perturbing the function of local proton gates, and result in an uncontrolled proton translocation across the antiporter module. The molecular consequences of disease mutations are discussed in the context of the proposed pumping mechanism.
Membrane-bound decarboxylases couple carboxylic acid decarboxylation to the transport of Na+ ions out of prokaryotic cells. The molecular mechanism of decarboxylase action is not yet known, which contrasts with the progress achieved in studying other primary ion pumps. Measuring decarboxylase activity is complicated by slow keto-enol tautomerization of the substrates during the assay. We found that HEPES exhibits anomalously high efficiency as a general acid catalyst for CH bond formation during the enol-to-ketone conversion of oxaloacetate. Accordingly, the addition of HEPES to the assay medium eliminated the contribution of tautomerization rate to measured decarboxylation rate. Using the dependence of oxaloacetate tautomerization rate and equilibrium on solvent properties and pH, we established that only the keto form of oxaloacetate is converted by Vibrio cholerae oxaloacetate decarboxylase. Steady-state kinetic measurements did not reveal cooperativity in oxaloacetate conversion and Na+ binding. The effects of ionophores (CCCP, valinomycin, and ETH157) on proton transport in pyranine-loaded membrane vesicles prepared from V. cholerae cells indicated that the proton required for the conversion of oxaloacetate to pyruvate is taken up from the cytoplasmic side of the membrane. Furthermore, the effects suggested that ΔpH generation is caused by secondary electrophoretic proton transport in exchange for Na+. These findings advance our understanding of the molecular mechanism of the decarboxylation-supported Na+ transport in bacteria.
Phycobiliproteins, particularly C-phycocyanin (CPC), serve as major light-harvesting complexes in cyanobacteria, exhibiting high efficiency in light-harvesting and energy transfer. Currently, the easy and large-scale acquisition of functional CPC remains a major challenge, primarily due to the requirement for precise, sequential covalent attachment of multiple phycocyanobilin (PCB) chromophores. In this study, by using a dual-promoter (T7 and araBAD) system to control the sequential binding of two PCB chromophores to β82 and β153, we successfully achieved the biosynthesis of β-CPC (λmax, absorption = 605 nm, λmax, emission = 644 nm) in E. coli, which can transfer energy from the β153-PCB to the β82-PCB. The assembly of α-CPC with PCB-β82, PCB-β153, and PCB2-β under identical conditions indicates that only PCB2-β, which covalently binds the two PCB chromophores, can assemble with α-CPC to form a complete CPC trimer (λmax, absorption = 617 nm, λmax, emission = 646 nm). The structure of the assembled trimer reveals that it adopts a typical phycobiliprotein fold, with multiple chromophores precisely arranged, exhibiting features highly similar to those of native CPC. Furthermore, we established a biosynthetic pathway for CPC trimers in E. coli. This system provides a powerful tool for engineering phycobiliproteins with various light-harvesting and energy transfer properties, facilitating future studies on artificial photosynthesis, light-harvesting antenna design, and the fundamental mechanisms of excitation energy transfer.
Photosystem II (PSII) catalyzes water oxidation into electrons, protons and dioxygen at its catalytic center, a Mn4CaO5 cluster, utilizing light energy. An amino acid residue D1-V185 in the D1 protein is located close to the Mn4CaO5 cluster, and plays a critical role in its catalytic function. In this research we purified PSII dimers from a D1-V185T mutant of Thermosynechococcus vestitus and analyzed its structure using low-damage cryo-electron microscopy (cryo-EM) at a resolution of 1.88 Å. The results revealed the presence of multi-conformations at the mutation site. Unlike the wild-type valine, which does not allow water molecules to be able to form hydrogen-bonds with it, both conformations of the mutant formed hydrogen bonds with nearby water molecules, which leads to rearrangement of the hydrogen bond networks in the O1 and Cl-1 channels. In conformation-A, the mutated Thr residue forms a hydrogen bond with a water molecule W6, which creates a new channel that bypasses the original O1 channel. Due to the hydrophilic OH group of Thr, the side-chain of D1-Glu189 was attracted and shifted toward the mutant Thr residue. In conformation-B, it forms a hydrogen bond with a water molecule W9 in the Cl-1 channel, bringing W9 closer and thereby disrupting the hydrogen bond network of the Cl-1 channel. In addition, multi-conformations of D2-K317, which is a ligand of Cl-1, were found in the mutant. These changes alter the environment surrounding the Cl-1 ion and Mn4CaO5, thereby affecting the PSII water-oxidation activity.
Regulation of proton motive force (pmf) via ATP synthase activity is a critical mechanism by which photosynthetic organisms maintain redox homeostasis and control the activation and inactivation of photoprotective responses under fluctuating light conditions. Here, we used time-resolved electrochromic shift measurements to investigate pmf dynamics in the C3 model plant Arabidopsis thaliana and the C4 model grass Setaria viridis. Our results reveal that ATP synthase is dynamically regulated during light fluctuations, but in Arabidopsis this regulation could not be explained by the established light-induced reduction of the CF₁γ subunit by thioredoxins, suggesting alternative control mechanisms. The PROTON GRADIENT REGULATION 5 (PGR5) protein, previously proposed to facilitate cyclic electron transport (CET) in plants and algae, also has a potential role in regulation of ATP synthase. We therefore investigated pmf dynamics, cytochrome f redox changes, and linear and cyclic PSI electron transport rates in WT and pgr5 knock-out mutants and revealed that while PGR5 was not required for CET, it was needed for downregulating ATP synthase under high irradiance in both species. Furthermore, in Arabidopsis disturbance of thiol redox regulation by addition of N-ethylmaleimide resulted in downregulation of ATP synthase conductivity in WT but not in pgr5 mutants, and PGR5 interacted with CF₁γ in planta. We suggest that PGR5 functions as a conserved thiol redox state dependent inhibitor of chloroplast ATP synthase under high light, contributing to pmf retention and photoprotection.
Photosynthesis in cyanobacteria relies on light capture by photosystem I (PSI), photosystem II (PSII) and the phycobilisome (PBS). Although these complexes are generally considered to be intermixed within the thylakoid membrane, several studies have suggested the presence of PSI- or PSII/PBS-enriched microdomains that may depend on environmental conditions. Here we applied cryo-Expansion Microscopy (cryo-ExM) to dark-adapted Synechocystis sp. PCC 6803 cells and achieved nanoscale resolution of thylakoid compartments and associated protein complexes. Cells were cryofixed, rehydrated at room temperature and physically expanded in a swellable hydrogel. By expanding cells 5.5-fold, we resolved individual thylakoid compartments in intact cells using confocal microscopy. Furthermore, immunolabeling allowed simultaneous localization of PSI, PSII and PBS within the expanded thylakoid network. Overall PSI, PSII, and PBS signals showed similar spatial distributions. However, PBS was excluded from the neck region between dividing cells, while PSI and PSII were present. These results establish cryo-ExM as a powerful method for visualizing cyanobacterial thylakoid membranes and mapping the distribution of key photosynthetic complexes, thereby complementing existing approaches for dissecting the spatial organization of photosynthesis.
Mitochondrial function is crucial for the regulation of energy metabolism, proton homeostasis, and stress adaptation in Saccharomyces cerevisiae. This study demonstrates the role of mitochondria in modulating cellular responses to varying extracellular pH (3.0, 5.0, 6.5) and glucose availability (0.5%, 2%). Results indicate that mitochondrial deficiencies in Δhap4 and ρ0 mutants selectively impair growth under acidic pH and 0.5% glucose conditions, whereas wild-type cells maintain pH-independent growth. Mitochondrial impairment redistributes intracellular H+ homeostasis regulation to plasma membrane and cytosolic H+-ATPases in a glucose- and pH-dependent manner, with ρ0 cells exhibiting maximal reliance on non-mitochondrial ATPases. The N, N'- dicyclohexylcarbodiimide (DCCD)-sensitive JH+ scales inversely with glucose availability, reflecting energy demand under nutrient limitation and acid stress. ρ0 cells exhibit the highest alcohol dehydrogenase activity to regulate the redox balance in response to non-functional mitochondria. The highest total H+-ATPase activity measured in ρ0 cells at pH 6.5 and 0.5% glucose conditions, combined with proton flux data, indicates the upregulation of plasma membrane and cytosolic ATPases activity for maintaining proton motive force and intracellular pH due to a complete loss of FoF1-ATPase contribution. These results pave the way for the construction of robust S. cerevisiae yeast strains to varying glucose and extracellular pH conditions.
The charge separation mechanism in spinach photosystem II core complexes (PSII-CC) and D1/D2/cytb559 reaction centers (D1D2-RC) was investigated using broadband femtosecond absorption spectroscopy in combination with kinetic modeling. The population dynamics of electronic states was analyzed based on decomposing the spectral-temporal matrices ΔA(λ,t) into contributions from excited states of chlorophyll (Chl) and difference spectra of the oxidized electron donor P680 and the reduced pheophytin acceptor PheoD1 obtained by the steady-state photoaccumulation technique. The results are discussed within the models, suggesting that in D1D2-RC at 279 K/77 K: (i) the predominant mechanism of primary charge separation is formation of the ChlD1+PheoD1- ion-radical pair in 3.6/7.6 ps followed by the hole transfer to P680 in 30/108 ps; (ii) the excitation energy transfer from ChlZ molecules to central RC pigments occurs within 19/28 ps, overlapping in time with the electron-transfer reactions; (iii) the free energy level of ChlD1+PheoD1- is higher than that of the relaxed exciton state by ∼30 meV, but the states become equally energetic during the ChlD1+ → P680 hole transfer. The charge separation kinetics in D1D2-RC at 77 K was found to be significantly heterogeneous. In PSII-CC at 279 K, (i) the apparent time of energy transfer from the excited antenna to RC is ∼60 ps, while the effective time of the ChlD1+PheoD1- formation in a small fraction of complexes with directly excited RC is comparable to that in D1D2-RC (3.7 ps); (ii) unlike D1D2-RC, the primary charge separation occurs with a decrease in free energy between RC⁎ and ChlD1+PheoD1-.
Photosynthesis constantly adapts to fluctuating light and temperature. Under high light, non-photochemical quenching (NPQ) protects the photosynthetic machinery by dissipating excess excitation energy as heat. With rising global temperatures and more frequent heat events, understanding how photosynthesis responds to combined light and temperature stress is critical. In vascular plants, NPQ is regulated by the protonation of Photosystem II subunit S (PsbS) and the enzymatic formation of zeaxanthin, but it remains unclear how temperature influences the complete NPQ response and the roles of these molecular components. Here, we analyzed NPQ in Arabidopsis thaliana WT and mutant genotypes affecting PsbS, zeaxanthin, or thylakoid membrane fluidity: npq1 (no zeaxanthin), npq2 (constitutive zeaxanthin), npq4 (no PsbS), PsbS-OE (overexpressed PsbS), and fad7fad8 (more rigid membranes). Plants were acclimated to 10 °C, 20 °C, 30 °C, or 40 °C for 30 min before measuring NPQ induction and relaxation. NPQ kinetics generally accelerated with increasing temperature, enabling faster responses to fluctuating light, and more absorbed energy was allocated to photochemistry rather than dissipation. Our results suggest that the concentration of PsbS is important to regulate this balance between photochemistry and NPQ. Full NPQ development and its temperature dependence required both PsbS and zeaxanthin. Arrhenius analysis of the NPQ induction and relaxation rates revealed that NPQ induction could be hindered by a rigid thylakoid membrane. These results demonstrate that NPQ is modulated by temperature, highlighting the importance of considering thermal effects on photoprotection when predicting plant performance under future climates.
Millisecond delayed light (DL) of bacteriochlorophylls was measured following rectangular light excitation of variable duration from intact purple photosynthetic bacteria with different electron donor configurations: the cytochrome-less mutant cycA of Rhodobacter sphaeroides (single turnover), wild-type Rubrivivax gelatinosus and Blastochloris viridis (multiple turnovers with bound tetraheme cytochrome), and the pufC mutant (loosely bound alternative donors). All strains exhibited similar dominant DL decay components (1-10 ms) but with markedly different amplitudes. In cycA, DL decayed much faster than the P+QA- → PQA charge-pair recombination (≈ 60 ms) and the saturation of its amplitude was delayed relative to prompt fluorescence (PF) by more than an order of magnitude relative to the rise time. Millisecond DL in cycA resulted from direct deactivation-type radiative recombination (via nuclear tunneling through P*) of a small subpopulation (α ≈ 2 × 10-6) of P+QA- states, as determined from the DL-to-PF intensity ratio. Strains capable of multiple turnovers exhibited ∼100-fold enhanced DL (α ≈ 10-4 in Rvx. gelatinosus), attributed to redox equilibrium between P/P+ and cytochrome c2+/c3+ that continuously repopulated P+QA-. This enhancement was modulated by proton-motive force (pmf), as demonstrated by sensitivity to protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Electron donor ferrocene quenched DL in all strains, consistent with perturbation of the redox equilibrium. Millisecond DL as an optical reporter of redox poise and membrane energization in intact photosynthetic bacteria offers valuable contribution to understanding energy dissipation pathways in bacterial photosynthesis and energization state of photosynthetic membranes.
The PSI-LHCI chlorophyll-protein complex is a nano-machine within the photosynthetic system in which a small subset of chlorophyll molecules absorbs light at energies lower than that of the reaction center (RC). Despite this energy mismatch, the low-energy chlorophyll forms (LES) efficiently transfer excitation energy (EET) to the RC, contributing to charge separation. At room temperature, more than 80% of the thermally equilibrated excitation population resides on LES, implying that photochemical trapping frequently involves energetically uphill EET. This raises a fundamental question: how can excitation reach the RC against an energy gradient? Here, a thermodynamic framework is developed to address this question. By combining known experimental observations with a coarse-grained analysis of excitation energy transfer, it is concluded that the additional energy required to overcome the energy gap between LES and the RC comes from thermal energy fluctuations in the chlorophyll environment, including the protein scaffold and membrane, which act as an active energy source. The estimated internal energy fluctuations of the amino acid residues coordinating LES chlorophylls exceed this energy gap, thereby making fluctuation-assisted transfer physically plausible. Within this viewpoint, the excited chlorophyll-protein system operates as a stochastic rectifier, selectively capturing thermal fluctuations to drive excitation towards the RC against an energy gradient. This work proposes a shift in perspective, from viewing environmental fluctuations as a passive background to recognizing them as an active thermodynamic resource enabling uphill excitation energy transfer.
Internodal cells of Characeae exposed to flickering spotted illumination mobilize long-range interchloroplast communications between the point of local light stress and non-treated cell areas. The signaling and functional coordination between immobile chloroplasts involves the export of excess products from light-stressed plastids, the lateral transport by the streaming cytoplasm, the entry of delivered substances to dimly lit recipient chloroplasts, and metabolic responses to the entered agents. There is indirect evidence that interchloroplast communications are mediated by two types of metabolites produced during photosynthetic electron transport: reducing agents such as NAD(P)H and the product of oxygen reduction H2O2 as the most stable ROS form. The involvement of these substances in intracellular signaling was tested in this study using local extracellular application of subnanomolar quantities of H2O2 and the cytoplasmic microinjection of NADH in combination with Microscopy-PAM chlorophyll fluorometry and confocal laser scanning microscopy. The pointed introduction of the above agents affected the actual and maximal chlorophyll (Chl) fluorescence yields (F' and Fm') in chloroplasts exposed to dim background light but had no effect in darkened cells. The Chl fluorescence changes induced by NADH and H2O2 featured opposite polarities, indicating the plastoquinone reduction (via segments of cyclic electron-transport pathways) and the development of non-photochemical quenching, respectively. The ability of externally applied H2O2 to move with the cytoplasmic flow has been revealed; it confirms that H2O2 can act in plant cells as a transportable signaling substance. The results provide evidence for participation of reducing substances and the oxidizing agent H2O2 in interchloroplast communications.
Photosystem I (PSI) converts light energy into chemical energy in photosynthesis, and forms supercomplexes with light-harvesting complexes (LHCI) in eukaryotes to enhance energy capture and transfer. Various numbers and organizations of both PSI core and LHCI subunits are observed in various organisms. A subgroup of haptophytes named coccolithophores play a major role in marine carbon cycle and CaCO3 production, and the light-harvesting antennas of them are named FCPs (fucoxanthin-chlorophyll a/c binding protein) because they bind chlorophyll c and fucoxanthin in addition to chlorophyll a. A structure of a large PSI-FCPI supercomplex containing 38 FCPI subunits has been reported from a coccolithophore Emiliania huxleyi recently (L. Shen et al., Science 389, eadv2132, 2025). Here we solved five cryo-electron microscopy (cryo-EM) structures of PSI-FCPI supercomplexes isolated from another coccolithophore Chrysotila roscoffensis with different detergents at resolutions ranging from 2.3 to 1.7 Å. These structures represent discrete PSI-FCPIs containing 1, 4, 6, 8 and 9 FCPI subunits, with FCPIs arranged in a modular fashion. Association of each FCPI module to the PSI core, as well as the arrangement of protein subunits and pigments, are revealed. Contributions of individual antenna modules to excitation energy transfer were calculated and compared with PSI-FCPI supercomplexes from other species of coccolithophores and haptophytes. These results pinpoint the assembly of stable PSI-FCPI supercomplexes in C. roscoffensis and provide insights into how antenna modules contribute to energy transfer in coccolithophores.
Aggregation of the main antenna complex of higher plants, Light-Harvesting Complex II (LHCII), is widely used as an in-vitro model for energy-dependent quenching (qE), yet fluorescence reduction in aggregates is frequently interpreted without a quantitative separation of intrinsic quenching from excitation-induced annihilation. Here, we address this ambiguity by directly correlating aggregate size, concentration, steady-state fluorescence intensity, and decay kinetics during controlled, incremental aggregation of isolated LHCII. By combining fluorescence correlation spectroscopy (FCS) with time-correlated single-photon counting (TCSPC) in a unified experimental framework, we monitored structural and photophysical changes in real time as detergent removal drives biphasic aggregation. We quantified the aggregate composition from the particle concentrations, enabling direct scaling of the absorption cross-section with aggregate size. The average fluorescence lifetime decreased semi-logarithmically with increases in hydrodynamic radius, whereas steady-state fluorescence intensities deviated strongly from this trend. Intensity-dependent measurements and steady-state kinetic modeling reveal that singlet-triplet annihilation (STA) emerges at moderate excitation intensities and rapidly becomes the dominant contributor to fluorescence quenching, even for relatively small aggregates. In contrast, intrinsic quenching increases more gradually with aggregate size. By quantitatively disentangling intrinsic excitation quenching from annihilation processes, this work demonstrates that STA can govern the apparent photophysical response of aggregated LHCII across excitation regimes commonly considered non-annihilating. The size-dependent mechanistic framework presented here provides a basis for distinguishing intrinsic quenching from annihilation effects in aggregation-based studies of photosynthetic antenna complexes.
Recently in these pages, a paper by Lynch appeared in response to a report showing that his numbers for biosynthetic costs (ATP demand) in cells are inflated, so much so that they would require E. coli to obtain >100 ATP per glucose and mitochondria to obtain >240 ATP per glucose. The inflated estimates trace to one factor: Lynch exclusively considers ATP demand and systematically neglects ATP supply—the essence of bioenergetics. Thermodynamics stipulate that a cell cannot grow if its ATP demands exceed its ATP supply. Here I compare Lynch's calculated ATP demands to laboratory measurements of the ATP supply that E. coli synthesizes during cell division. The results bear out my case, and leave no doubt: Lynch's calculations require E. coli to synthesize ∼120 ATP per glucose, which is thermodynamically impossible. As a consequence, his demand-only ‘energetic’ attacks on mitochondria and endosymbiosis in evolution are baseless.
Glycerophosphate shuttle, an important crossroad between oxidative phosphorylation system, glycolysis and lipid metabolism, consists of the rate-limiting mitochondrial glycerol-3-phosphate dehydrogenase (GPD2) and the cytosolic dehydrogenase (GPD1). GPD2 level is relatively high in islet beta-cells, spermatozoa and neurons, required abruptly rapid periodic ATP consumption, as well as in rapidly growing normal tissues during neonatal period and many cancers. According to the computational model developed in the present work, the glycerophosphate shuttle should be significantly activated by the outer membrane potential (OMP) generated by the VDAC1,2-hexokinase complexes of mitochondrial outer membrane. This is due to the capture of cytosolic glycerol-3-phosphate2- into the mitochondrial intermembrane space by the positive OMP, thus increasing its local concentration near GPD2. The predicted acceleration is most significant at relatively high Km of GPD2 for glycerol-3-phosphate2- and strongly modulated by the VDAC's voltage-gating properties. In general, OMP generated by the VDAC1,2-hexokinase complexes might play a crucial role in the above-mentioned crossroad, converting it into the "electrical metabolic crossroad". The suggested electrical deviation of glycolysis towards the mitochondrial GPD2, as a tool for the metabolic shift to an accelerated aerobic glycolysis without an inhibition of mitochondrial respiration, highlights this metabolic switching as one of the possible options of the Warburg effect.
According to the modern classification of NiFe hydrogenases, the HydSL hydrogenase of Thiocapsa bogorovii is assigned to subgroup 1e, the so-called isp-type hydrogenases. This subgroup is proposed to unite hydrogenases involved in sulfur respiration; however, direct experimental evidence supporting this function remains limited. In this study, we isolated a protein complex containing the HydSL hydrogenase from T. bogorovii that exhibits high activity in the H₂ + S0 → H₂S reaction. Under native electrophoresis, the complex migrated as a single band, whereas SDS-PAGE resolved it into seven bands, four of which matched the molecular masses of HydS, Isp1, Isp2, and HydL proteins. Incubation of T. bogorovii cells in a hydrogen atmosphere in darkness and in the presence of elemental sulfur resulted in increased expression of the hydS, hydL, isp1, and isp2 genes encoding the subunits and partner proteins of the complex. The elevated transcript levels correlated with increased cellular activity in the reduction of elemental sulfur to hydrogen sulfide. These observations indicate that the HydSL-containing protein complex may participate in sulfur respiration in T. bogorovii under dark, anaerobic conditions in the absence of oxygen and fermentable substrates. Based on these results and literature data, we propose a putative mechanism for the complex, consistent with its designation as a sulfhydrogenase.
