Biochimica et Biophysica Acta (BBA) - Bioenergetics

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

Conflicting roles have been proposed for the E. coli protein RatA. Initially described as a ribosome targeting toxin, a later report pronounced it the bacterial homologue to the inner mitochondrial membrane protein Coq10. Coq10 proteins are conserved from prokaryotes to human and implicated to serve a lipid chaperone role in the biosynthesis of ubiquinone, a crucial electron carrier during aerobic respiration. We recently identified that the contradictory results published for RatA can be attributed to a mis-annotation of the gene in the reference genome. Here, we further elucidate the molecular function of RatA. We clarify that RatA is not a toxin but serves as a lipid shuttle for ubiquinone from its cytosolic biosynthesis complex to the inner membrane. Furthermore, we show that the loss of RatA results in an impaired, but not abolished electron transport chain and demonstrate broad metabolic adaptations of the cells as a consequence. Therefore, we propose to rename RatA to UbiM to reflect its function and to be in accordance with the naming convention of other ubiquinone biosynthesis proteins.

Sulfide:quinone oxidoreductase (SQR) is a critical enzyme that maintains sulfur metabolism by oxidizing sulfide to supersulfides, currently defined as sulfur metabolites with six valence electrons and no charge that are covalently catenated with other sulfur atoms and excludes disulfides. While SQR is known to contribute to mitochondrial electron transport, its physiological impact on systemic energy metabolism and longevity remains largely undefined. In this study, we investigated the role of SQR in mitochondrial bioenergetics and aging using SQR-deficient Schizosaccharomyces pombe ({Delta}hmt2) and a mitochondria-selective SQR-deficient (Sqrdl{Delta}N/{Delta}N) mice model. Functional analysis demonstrated that{Delta} hmt2 grew normally in glucose but not in glycerol, indicating impaired mitochondrial respiration. It showed reduced membrane potential, ATP, and lifespan. Consistent with the yeast findings, Sqrdl{Delta}N/{Delta}N mice exhibited accumulated levels of hydrogen sulfide and persulfides, and demonstrated impaired mitochondrial energy metabolism. Furthermore, supersulfide donor supplementation selectively conferred lifespan extension in wild-type yeast, but not in SQR-deficient strain, and similarly improved mitochondrial function exclusively in wild-type mouse embryonic fibroblasts, with no benefit observed in SQR-mutant counterparts. Together, our findings demonstrate that mitochondrial SQR plays an essential role in sulfur respiration, critically supporting mitochondrial function and organismal longevity across eukaryotes. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/716515v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@d834forg.highwire.dtl.DTLVardef@127dc7dorg.highwire.dtl.DTLVardef@1fccb8eorg.highwire.dtl.DTLVardef@197e910_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeveloped an SQR-deficient S. pombe ({Delta}hmt2) model that exhibits sulfur metabolism, mitochondrial dysfunction, and shortened chronological lifespan C_LIO_LISulfide and supersulfide donors prolong yeast lifespan in a SQR-dependent manner C_LIO_LIMitochondrial SQR is essential for membrane potential formation and ATP production in yeast and mammals C_LI

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H2, making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O2 sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani, has exhibited exceptional O2-stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O2-induced degradation by formation of the Hinact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe]H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the Hinact and Htrans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydAPDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fed center of [2Fe]H, thereby reducing Hinact accumulation. Moreover, as-isolated ToHydAPDT predominantly exhibits the Hhyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe]H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

Carbon monoxide dehydrogenases (CODHs) are metalloenzymes central to microbial CO metabolism and CO2 fixation. We report the heterologous production and characterisation of Clostridium pasteurianum BC1 CODH-III (CpBC1CODH-III), from the phylogenetic clade E, co-expressed with its maturation machinery CooCTJ. CpBC1CODH-III shows moderate CO oxidation (150 U/mg) and CO2 reduction (0.568 U/mg) activities. Electron paramagnetic resonance (EPR) spectroscopy under varying redox conditions identified a rhombic signal at g {approx} 2.0, characteristic of reduced B-clusters, and a C-clusters at different stages (g {approx} 1.75, g {approx} 1.72), indicative of a bound CO2. Investigation of maturation effects showed that co-expression of CooCTJ stabilised CpBC1CODH-III production, but did not enhance maximum activity, which was primarily influenced by nickel availability. Comparative operon analysis with the well-studied clade F Rhodospirillum rubrum CODH (RrCODH) revealed high structural similarity in CODH and CooC, but significant divergence in CooJ, with conserved metal-binding regions identified via AlphaFold3 modelling and dot plot analysis. CpBC1CODH-III represents a unique example of a clade E CODH within a clade F genomic context, demonstrating intrinsic robustness in maturation and activity

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. Here, we use site-directed mutagenesis to study the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Mutations at this position change the biochemical properties (KM for CO, product inhibition constant, catalytic bias...), and increase the resistance of the enzyme to the inhibitor O2, showing that isoleucine 563 indeed lines the gas channel. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far. We show that the size of the introduced amino acids is less important than their flexibility - along with the size of the cavity formed near the active site in the channel. We also conclude that O2 access to the active site cannot be slowed down without also affecting CO diffusion. This tradeoff will have to be considered in further attempts to use site-directed mutagenesis to make CODHs more O2 tolerant.

A method to measure telomere length in S. cerevisiae was developed based on bioluminescence resonance energy transfer (BRET). The system uses energy transfer between a luciferase-Rif2 fusion protein and fluorescently tagged Rap1. The study demonstrates that the BRET ratio correlates with the Rap1/Rif2 complex at the telomeres and thus the availability of telomeric Rap1 binding sites. This enables the measurement of telomere length in living cells. The system was able to reproduce reported deviations in telomere length in mutants lacking telomere length regulators, cells treated with telomere length modifying compounds and strains expressing inducible telomerase. The BRET ratio linearly correlated with the average number of telomeric nucleotides derived from long-read sequencing data using a novel algorithm for telomere length calculation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711003v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1850c4dorg.highwire.dtl.DTLVardef@1ead295org.highwire.dtl.DTLVardef@1a76358org.highwire.dtl.DTLVardef@6b3183_HPS_FORMAT_FIGEXP M_FIG C_FIG

Long-range protein electron transfer (ET) often depends on tryptophan and tyrosine residues acting as radical relay sites. For example, cytochrome c peroxidase (CcP) generates a W191*+ radical to increase ET from cytochrome c (Cc) to the active center. W191 substitution to Tyr reduces ET rates, but introduction of an adjacent general base at position 232 (as Glu or His) recovers activity. E232 fluorination shifts the ET pH dependence to lower values, verifying that a hydrogen bond elevates the Y191* formal potential for effective ET. Photoinitiated ET between Zn-porphyrin (ZnP) CcP (ZnCcP) and Cc also depends on activating Y191 with a basic residue, but through a different mechanism than for the peroxide-driven system. In ZnCcP, pH dependencies and solvent isotope effects indicate that proton-coupled electron transfer to the basic residue and ZnP*+, respectively, facilitate Y191* formation. Replacing Cc with the irreversible oxidant [Co(NH3)5Cl]2+ isolates distinct protein radicals for characterization by Electron Paramagnetic Resonance (EPR) spectroscopy. Radical distributions reveal that W191*+ lies [~]15 mV in potential below ZnP*+ and that the two radicals exchange on a slow time scale despite their close separation. Remarkably, ZnCcP Y,G191:E,H232 variants propagate radicals differently to peripheral sites depending on the nature of the 232 residue. QM/MM calculations support radical exchange between ZnP*+/Trp*+ and the importance of a hydrogen bond to Y191* for maintaining a high potential to oxidize peripheral donors. These resolved reactivity patterns of CcP/ZnCcP have general relevance for engineering proton management to separate and migrate charge in proteins and potentially other molecular systems.

Oxidative phosphorylation is the most efficient way of generating ATP in respiring cells. As high energy electrons are the major source of reactive oxygen species their production needs to be carefully calibrated. In most organisms, NADH dehydrogenase serves as the primary source and gateway of electrons. This complex is responsible for oxidizing NADH to NAD+, which liberates two electrons that are then fed into the respiratory chain. In the Gram-positive model bacterium, Bacillus subtilis, a transcription factor (Rex) is utilized to monitor the rise in NADH level and subsequently increase the production of the NADH dehydrogenase Ndh. Thus, the generation of electrons through this pathway is tightly regulated. In this report, we reveal the presence of another independent mechanism to moderate Ndh activity involving a previously uncharacterized protein, YfhS. Additionally, we present the first experimental evidence showing that the functional NADH dehydrogenase is a two-protein complex comprised of a membrane-associated YjlC and the enzyme Ndh. We find that absence of YfhS leads to cell morphology and growth defects that are corrected by spontaneous mutations in ndh. We note that increased production of NADH dehydrogenase complex proteins by itself is not detrimental. However, strikingly, it is lethal in a strain lacking yfhS. These results reveal that YfhS is an important moderator of NADH dehydrogenase activity. We also demonstrate that YfhS and YjlC are interaction partners. A model developed based on our data indicates that YfhS is an important regulator of intracellular NADH concentration. Compounds that target specific microbial (Type II) NADH dehydrogenase, which is absent in human mitochondria, are considered promising drug candidates to help address the threat posed by antibiotic-resistant bacteria. Overall, our data unveiling the importance of YfhS and YjlC in controlling Ndh activity could be harnessed for the development of new therapeutics.

Many biochemical processes are dependent on the presence or absence of molecular oxygen (O2). Palladium-tetrapyrrol derivatives can be used to measure O2-concentrations and O2-turnover during biochemical reactions and microbial growth in standard microtiter plates (MTPs). Palladium(II)-5,10,15,20-(tetrapentafluorophenyl)-porphyrin (1; CAS 72076-09-6) and Palladium(II)-5,10,15,20-(tetraphenyl)tetrabenzoporphyrin (2; CAS 119654-64-7) are introduced with this study. Spectral analyses of both compounds revealed that fluorescence quenching by O2 is not evenly distributed throughout all wavelengths and can therefore be used ratiometrically. Experimentally determined fluorescence lifetimes are around 500 {micro}s and 300 {micro}s for 1 and 2, respectively. A simple protocol is disclosed, how to immobilize the indicators on the bottom of MTP wells to give clear transparent dye doped polymer layers. We propose a straightforward procedure of how fluorescence data can be processed and calibrated in terms of O2 concentrations. Diverse applications are demonstrated and discussed, which include oxygen consumption and production by microorganisms as well as by enzymatically catalysed biochemical reactions. Various aspects are critically considered, as there are e.g. the dependence of O2 solubility on temperature and salinity, the diffusion of O2 across diverse phase boundaries, the unwanted O2 ingress into the reaction volume, the oxygen binding capacity of the MTP plastic material and the pH-dependence of the sensor layer. The findings and methods presented here open up a broad variety of high throughput assays involving changes of dissolved O2 as measurands for biochemical and biological activity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/718663v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@4daa4dorg.highwire.dtl.DTLVardef@e7ab8aorg.highwire.dtl.DTLVardef@1af1149org.highwire.dtl.DTLVardef@97fea5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

How did phosphate become the universal energetic currency of life? Traditional approaches to phosphorylation in early evolution studies entail oven drying, non-aqueous solvents, dangerously reactive forms of phosphorus, or other non-physiological conditions. With microbial physiology as a vade mecum, we have recently found that phosphite, HPO32-, which is enzymatically oxidized by many microbes and which naturally occurs in serpentinizing hydrothermal vents, will readily phosphorylate ribose, glucose, glycerol, serine, AMP, creatine and acetate to generate phosphoester, phosphoanhydride and acylphosphate bonds in hours to days at 25-100{degrees}C in pure alkaline water. These reactions are thermodynamically favourable because anoxic phosphite oxidation to phosphate and H2 is highly exergonic, but they do not proceed without catalysts. The most effective catalyst yet identified is a nanoparticular form of a shiny metal: zero-valent (native, or elemental) palladium (Pd0). Native palladium, like phosphite, also naturally occurs in serpentinizing hydrothermal vents, as do other native platinum group elements (PGE), including Pt, Rh, Ru and Ir. Here we test those PGE as catalysts of phosphite oxidation and phosphorylation. Though all metals tested readily oxidize phosphite, only Pd0 efficiently catalyzes phosphorylation, generating phosphorylated products at concentrations often equal to their physiological concentrations in growing Escherichia coli cells. Metaphosphate is a possible reaction intermediate. In phosphorylation reactions via phosphite oxidation (DG0'= -46 kJ{middle dot}mol-1), a portion of the energy released is conserved in phosphorylated products, as in biological energy conservation. A natural environment and energy-conserving thermodynamics implicate these facile aqueous phosphorylating reactions in the origin of bioenergetics.

Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.

This study investigates the potential of the garden rhizosphere as a source of electrochemically active bacteria (EAB) for operating microbial fuel cells (MFCs). We evaluated a diverse array of garden flora, including vegetables (lettuce, Chinese cabbage), flowering plants (August lily, peppermint), and woody species (pine, oak, ginkgo, and bush clover). Among the tested groups, MFCs inoculated with peppermint and ginkgo rhizosphere microbiotas exhibited the highest current densities within their respective categories, significantly outperforming control groups without plant components. 16S rRNA gene microbial community analysis revealed that the initial rhizosphere environment acts as a decisive selective pressure, shaping distinct anode biofilms based on plant types (herbaceous vs. woody). Despite these structural differences in microbial assembly, high current generation was achieved in both peppermint and ginkgo systems, suggesting a high degree of functional redundancy within the rhizosphere-derived consortia. These findings demonstrate that various garden ecosystems can serve as robust biological reservoirs for MFC operation, where diverse microbial configurations are capable of sustaining efficient bio-electrochemical energy conversion.

The evolutionary transition of the green plant lineage (Viridiplantae) from aquatic environments to terrestrial habitats required unprecedented adaptations of cellular metabolism to severe environmental stressors, including desiccation, high irradiance, and rapid temperature fluctuations. Redox regulation, mediated by oxidation and reduction of reactive cysteine residues (RCys), plays a crucial role in translating environmental fluctuations into rapid cellular responses. Although comparative genomics has revealed expansions in multiple cellular systems preceding terrestrialization, the evolutionary history of redox-regulated protein networks remains elusive. This work integrated large-scale phylogenomic reconstructions across 37 Viridiplantae species with five independent Arabidopsis thaliana redox proteomics datasets to trace the evolutionary trajectory of RCys. The analysis showed that the ancestral core, consisting of plastid-localized regulatory cysteines, was already established at the base of the green lineage. Furthermore, an expansion driven by gains of RCys via amino acid replacements within pre-existing proteins occurred in the common ancestor of Zygnematophyceae and land plants. These findings suggest that a targeted incorporation of thiol-based regulatory switches provided early land plant ancestors with enhanced protein functional plasticity necessary to cope with the challenges of terrestrial environments. HighlightsO_LIThe foundational plastid-localized redox core was established at the root of Viridiplantae. C_LIO_LINovel regulatory switches were integrated into conserved machinery via amino acid replacement. C_LIO_LIA punctuated burst of redox innovation at Zygnematophyceae and Embryophyta last common ancestor preceded plant terrestrialization. C_LIO_LIRedox acquisition rates declined sharply following the successful colonization of land. C_LI

Invertebrate vision relies on bistable visual pigments flipping upon photon absorption between rhodopsin and metarhodopsin states. In living butterflies, the UV-VIS absorption spectra of rhodopsin and metarhodopsin, respectively with 11-cis and all-trans isomers of 3-hydroxy-retinal (A3) chromophore, can be conveniently recorded from the eyeshine, the light reflected from the compound eye after passing twice through the light-guiding rhabdoms. * Here, a microscope coupled with a broadband LED source and a microspectrometer was used to record photorelaxations reported in eyeshine reflection spectra. Fitting temporal exponential relaxations to log-reflectance arrays yielded transient and baseline spectra that are analogous to absorbance difference and sum, respectively. Both types of spectra were subjected to singular value decomposition and to fitting of templated visual pigment absorption spectra. * The compound eye of the high brown fritillary Fabriciana adippe was exposed to a series of second-long broadband light pulses, causing photorelaxations with time constants between 40 and 120 ms that led to 80% metarhodopsin in equilibrium. The transient and baseline spectra were fitted with pigment templates, estimating the alpha peak wavelength 547-552 nm for rhodopsin and 496-501 nm for metarhodopsin. The metarhodopsin to rhodopsin alpha peak absorbance ratio 1.25-1.35 is consistent with the isosbestic wavelength at 530 nm. The second isosbestic wavelength indicates that rhodopsin beta (UV) peak absorbs more strongly than metarhodopsin below 405 nm. * Baseline spectra, which were not explicitly analysed in previous studies, enable concatenation of exposures, monitor long-term changes of pigment, and enhance the estimation of beta peak parameters. * The method can be directly used in many butterflies and could be adapted to other insects, particularly fruitflies, facilitating studies of the relation between the visual pigment spectra and the opsin sequences. Spectroscopic results can be complemented with physiologically measured photoreceptor spectral sensitivity datasets and analysed with the same global fitting procedure.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Artemisinin is an effective antimalarial drug sourced from Artemisia annua, but its low and variable yields require enhancement either semi-synthetically or in-planta to meet the global demand for treatment. Though essential enzymes have been identified in the artemisinin biosynthetic pathway, including an essential Cytochrome P450 monooxygenase (CYP71AV1), there are still many unknowns. Cytochrome P450 reductase 1 (herein, AaCPR1), has been experimentally confirmed as an electron transfer partner for CYP71AV1 in its three step oxygenation of key artemisinin precursors. However, the recent discovery of a highly related CPR, herein AaCPR2, introduces the possibility that another, potentially more catalytically favourable interaction, could exist for CYP71AV1. Therefore, enzyme kinetics and differential scanning fluorimetry (DSF) were used in the characterisation of both AaCPR1 and AaCPR2 to determine the existence and source of their catalytic differences. Tested enzyme activity under cytochrome c and NADPH concentrations revealed that AaCPR1 had lower Km and higher kcat/Km values, while AaCPR2 had higher Vmax and kcat values. This suggests that AaCPR1 is more effective at reducing cytochrome c when substrate conditions are limiting, whereas AaCPR2 is more effective than AaCPR1 at reducing cytochrome c when substrate conditions are saturating. This implies a functional partitioning of the two enzymes on the basis of substrate availability. The DSF results provided deeper insight into the different protein-ligand interactions between the two enzymes. AaCPR2 reached lower maximum melting temperatures across all tested conditions, whereas AaCPR1 had higher maximum melting temperatures. Thus, AaCPR1 exhibits higher thermal stability and has a higher temperature threshold than AaCPR2. This contributes to the notion that the AaCPRs are functionally divergent also on the basis of temperature. The cumulative differences in melting behaviour between the two enzymes led to the hypothesis that AaCPR1 and AaCPR2 exhibit different domain motions that may lead to preferential catalysis for one redox partner over another. This was further supported by the prediction of a highly variable loop region between the two enzymes at the connecting domain just after the flexible hinge. If such loops are highly mobile, as predicted, then the residue differences therein could provide a bio-structural basis for the kinetic and thermal/biophysical differences observed between AaCPR1 and AaCPR2. These data support that AaCPR1 and AaCPR2 possess fundamental biophysical differences despite their high degree of relatedness. Ultimately, these differences suggest differential metabolic functions of the two enzyme in artemisinin biosynthesis and/or other important secondary metabolic processes.

The origin of eukaryotes is a key event in the evolution of cellular life hypothesized to involve a symbiotic integration between a member of the Asgard archaea and the Alphaproteobacteria. Recent work has provided evidence for additional genetic input from other prokaryotes to the eukaryotic proteome yet the extent and sources of these contributions remain debated. Here we aimed to further resolve the prokaryotic origins of eukaryotic genes to inform our understanding of eukaryogenesis. Specifically, we developed a phylogenetic framework to investigate the origins of eukaryotic gene families associated with metabolism and informational processing for comparison. We found that informational processing genes were predominantly derived by archaea whereas eukaryotic metabolism is highly chimeric in its origin. In contrast to previous studies, we report a substantial number of archaeal origins of diverse metabolic enzymes including key metabolic regulators. This highlights an overlooked participation of archaeal metabolism and pinpoints potential metabolic integrations during eukaryogenesis. Apart from the alphaproteobacterial contributions to the eukaryotic metabolism, we found an additional dominant phylogenetic signal of genes potentially derived from Myxococcota, especially for gene families associated with lipid metabolism. By systematically analysing the origins of eukaryotic metabolism, this research offers novel insights into the origin of eukaryotic membranes and refine our current models for the origin of the eukaryotic cell.