Biochimica et Biophysica Acta (BBA) - Bioenergetics

The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation.

Flash-induced absorption changes in the Soret region, which originate from the [PD1PD2]+ state, the chlorophyll cation radical formed upon Photosystem II (PSII) excitation, were investigated in Mn-depleted Photosystem II. In wild-type PSII from Thermosynechococcus elongatus, the [PD1PD2]+-minus-[PD1PD2] difference spectrum shows a main negative feature at 434 nm and a smaller negative feature at 446 nm [Boussac et al. Photosynth Res (2023), https://doi.org/10.1007/s11120-023-01049-3]. While the main feature at 434 nm is associated with PD1+ formation, the origin of the dip at 446 nm remains to be identified. For that, we have compared the [PD1PD2]+-minus-[PD1PD2] difference spectra from the PsbA3/H198Q PSII mutant in T. elongatus and D2/H197A PSII mutant in Synechocystis sp. PCC 6803 with their respective wild type strains. By modifying the PD1 axial ligand with the H198Q mutation in the D1 protein in T. elongatus, the contribution at 434 nm was shifted to 431 nm, while the contribution at 446 nm was hardly affected. In Synechocystis sp. PCC 6803, by modifying the PD2 axial ligand with the H197A mutation in the D2 protein, the contribution at 446 nm was downshifted by [~] 3 nm to [~] 443 nm, while the main contribution at 432 nm was only slightly shifted upwards to 433 nm. This result suggests that the bleaching seen at 446 nm involves PD2. This could reflects a change in the [PD1+PD2]{longleftrightarrow}[PD1PD2+] equilibrium or a more complex mechanism.

The effects of TyrD*, TyrZ*, and QA*- radical formation on the absorption spectrum in the Soret region were studied in Mn-depleted Photosystem II at pH 8.6 (in order to be in the TyrD state after dark adaptation). Flash-induced difference spectra were recorded in several PSII samples from: i) Thermosynechococcus vestitus (formerly T. elongatus), ii) Synechocystis sp. PCC 6803, iii) Chroococcidiopsis thermalis PCC 7203 grown under far-red light, and iv) Acaryochloris marina. In the case of T. vestitus, mutants D1/H198Q, D1/T1789H, D2/I178H, and D2/Y160F, with PsbA1/Q130 instead of PsbA3/E130, were also studied for possible contributions from PD1, ChlD1, ChlD2, and PheD1, respectively. For a possible contribution from PD2, the D2/H197A mutant was studied in S. 6803. While PD1 is clearly the species whose spectrum is blue-shifted by [~]3nm in the presence of QA*-, as has already been well documented in the literature, the species whose spectra shift upon the formation of TyrD* and TyrZ* remain to be clearly identified, as they appear different from PD1, PD2, PheD1, ChlD1, and ChlD2, as concluded by the lack of different light-induced difference spectra in the mutants listed above. Although we cannot rule out a weak effect, considering the accuracy of the experiments, it is proposed that other pigments, such as antenna Chl and/or Car, near the reaction center are involved. Additionally, it is shown that: i) there is no proton release into the bulk upon the oxidation of TyrD at pH 8.6, and ii) the rearrangement of the electrostatic environment of the pigments involved in the light-induced difference spectra in the samples studied, upon the formation of TyrD*, TyrZ*, and QA*-, likely occurs differently from both a kinetic and structural perspective.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of photo-activated form, OCPR. The most recent one suggests that three picosecond-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state which has the longest reported lifetime of a few to tens of picoseconds is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photo-dynamics of the OCP from Synechocystis PCC 6803, carried out using Visible-NIR femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photo-activation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S[~], living a few tens of picoseconds and formed only upon 470 nm excitation. Since neither the P1 quantum yield nor the photo-activation speed over hundreds of seconds vary under green and blue continuous irradiation, this S[~] species is unlikely to be involved in the photo-activation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on excited state photo-physical properties. Differences in spectral signatures and lifetimes of the different excited states were observed, at variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for careful consideration of the excitation power and His-tag position when comparing the photo-activation of different OCP variants, and beg to revisit the notion that S* is the precursor of photoactivated OCPR. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/474187v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1064473org.highwire.dtl.DTLVardef@77e3c7org.highwire.dtl.DTLVardef@10b1e0eorg.highwire.dtl.DTLVardef@e24977_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Qy and Bx excitation energy transfer (EET) in the minor light harvesting complex CP29 (LHCII B4.1) antenna complex of Pisum sativum was characterized using a computational approach. We applied Forster theory (FRET) and the transition density cube (TDC) method estimating the Coulombic coupling, based on a combination of classical molecular dynamics and QM/MM calculations. Employing TDC instead of FRET mostly affects the EET between chlorophylls (Chls) and carotenoids (Crts), as expected due to the Crts being spatially more challenging for FRET. Only between Chls, effects are found to be small (about only 0.1 EET efficiency change when introducing TDC instead of FRET). Effects of structural sampling were found to be small, illustrated by a small average standard deviation for the Qy state coupling elements (FRET/TDC: 0.97/0.94 cm-1). Due to the higher flexibility of the Bx state, the corresponding deviations are larger (FRET/TDC between Chl-Chl pairs: 17.58/22.67 cm-1, between Crt-Chl pairs: 62.58/31.63 cm-1). In summary, it was found for the Q band that the coupling between Chls varies only slightly depending on FRET or TDC, resulting in a minute effect on EET acceptor preference. In contrast, the coupling in the B band spectral region is found to be more affected. Here, the S2 (1Bu) states of the spatially challenging Crts may act as acceptors in addition to the Chl B states. Depending on FRET or TDC, several Chls show different Chl-to-Crt couplings. Interestingly, the EET between Chls or Crts in the B band is found to often outcompete the corresponding decay processes. The individual efficiencies for B band EET to Crts vary however strongly with the chosen coupling scheme (e.g., up to 0.29/0.99 FRET/TDC efficiency for the Chl a604/neoxanthin pair). Thus, the choice of coupling scheme must involve a consideration of the state of interest.

Purple non-sulfur bacteria (PNSB) are metabolically versatile microorganisms that inhabit diverse environments by taking advantage of a remarkably efficient photosynthetic machinery. In this work, we describe the creation of a workflow for experimental studies that aim to compare the energy transfer mechanisms occurring within structural variants of the light-harvesting complex 2 (LH2) of PNSB. Through the creation of a library of LH2 variants using site-directed mutagenesis, we engineered proteins with different spectral properties to be expressed in a LH2-defficient mutant of the model PNSB Rhodobacter sphaeroides. We validated this approach by reproducing a previously described mutant exhibitng a blue-shift, in addition to identifying a novel mutant exhibiting a red-shift of the B850 absorption peak. We characterised the fluorescence lifetime of the purified LH2 spectral variants in vitro, and performed a bacterial growth assay to assess the fitness of the LH2 variants in vivo under oversaturating light conditions. Our results suggest that the LH2s variants expressed by PNSB in nature reflect the intricate tunning of their quantum properties not towards the fastest energy transfer but towards the optimum light-harvesting efficiency which is defined by diverse environmental factors. Statement of significanceIn this work we report a platform for the systematic investigation of the mutational landscape of light harvesting complexes forming part of the photosynthetic machinery of purple non-sulfur bacteria. By conducting directed evolution of selected residues of the light-harvesting antenna (LH2) of Rhodobacter sphaeroides we identified a novel mutant with distinct and red-shifted spectral properties. We characterised the energy transfer dynamics of this and a previous characterised mutant and demonstrated that the new variant confers a phenotypic growth advantage when cultured with high-intensity light. Our findings offer new insights into the mechanisms of light capture and energy transfer also bridging the in vitro observations with quantifiable fitness advantages under the conditions tested.

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). Specialized Type-1 NDH complexes function as a part of this mechanism to couple photosynthetic energy generated by redox reactions of the electron transport chain (ETC) to CO2 hydration. This active site of CO2 hydration incorporates an arginine side chain as a Zn ligand, diverging from the typical histidine and/or cysteine residues found in standard CAs. In this study, we focused on mutating three amino acids in the active site of the constitutively expressed NDH-14 CO2 hydration complex in Synechococcus sp. PCC7942: CupB-R91, which acts as a zinc ligand, and CupB-E95 and CupB-H89, both of which are in close interaction with the arginine ligand. These mutations aimed to explore how they affect the unusual metal ligation by CupB-R91 and potentially influence the unusual catalytic process. The most severe defects in activity among the targeted residues are due to a substitution of CupB-R91 and the ionically interacting E95 since both proved essential for the structural stability of the CupB protein. On the other hand, CupB-H89 mutations show a range of catalytic phenotypes indicating a role of this residue in the catalytic mechanism of CO2-hydration, but no evidence was obtained for aberrant carbonic anhydrase activity that would have indicated uncoupling of the CO2-hydration activity from proton pumping. The results are discussed in terms of possible alternative CO2 hydration mechanisms.

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a {gamma}-subunit redox domain which contains a cysteine couple and interacts with the torque-generating {beta}DELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, {gamma}-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient [Formula]. Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the [Formula] present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra [Formula] generated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Fo {gamma}-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the {gamma}-disulfide. The obtained [Formula] -dependent CF1Fo activity profile in the two {gamma}-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Graber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two {gamma}-redox state were similar at high [Formula]. In the presence of the {gamma}-dithiol, similar rates were obtained at a ~45 mV lower [Formula] value compared to the oxidized state, which closely resembled the energetic gap of 0.7 {Delta}pH units reported in vitro.

The acclimation of cyanobacteria to iron deficiency is crucial for their survival in natural environments. In response to iron deficiency, many cyanobacterial species induce the production of a pigment-protein complex called IsiA. IsiA proteins associate with photosystem I (PSI) and can function as light-harvesting antennas or dissipate excess energy. They may also serve as Chl storage during iron limitation. In this study we examined the functional role of IsiA in cells of Synechocystis sp. PCC 6803 grown under iron limitation conditions by measuring the cellular IsiA content and its capability to transfer energy to PSI. We specifically test the effect of the oligomeric state of PSI by comparing wild-type (WT) Synechocystis sp. PCC 6803 to mutants lacking specific subunits of PSI, namely PsaL/PsaI ({Delta}psaL mutant) and PsaF/PsaJ ({Delta}FIJL). Time-resolved fluorescence spectroscopy revealed that IsiA formed functional PSI3-IsiA18 supercomplexes, wherein IsiA effectively transfers energy to PSI on a timescale of 10 ps at room temperature - measured in isolated complexes and in vivo - confirming the primary role of IsiA as an accessory light-harvesting antenna to PSI. However, a significant fraction (40%) remained unconnected to PSI, supporting the notion of a dual functional role of IsiA. Cells with monomeric PSI under iron deficiency contained only 3-4 IsiA complexes bound to PSI. Together the results show that IsiA is capable of transferring energy to trimeric and monomeric PSI but to varying degrees and that the acclimatory production of IsiA under iron stress is controlled by its ability to perform its light-harvesting function.

Photosystem II uses water as the ultimate electron source of the photosynthetic electron transfer chain. Water is oxidized to dioxygen at the Oxygen Evolving Complex (OEC), a Mn4CaO5 inorganic core embedded in the lumenal side of PSII. Water-filled channels are thought to bring in substrate water molecules to the OEC, remove the substrate protons to the lumen, and may transport the product oxygen. Three water-filled channels, denoted large, narrow, and broad, that extend from the OEC towards the aqueous surface more than 15 [A] away are seen. However, the actual mechanisms of water supply to the OEC, the removal of protons to the lumen and diffusion of oxygen away from the OEC have yet to be established. Here, we combine Molecular Dynamics (MD), Multi Conformation Continuum Electrostatics (MCCE) and Network Analysis to compare and contrast the three potential proton transfer paths during the S1 to S2 transition of the OEC. Hydrogen bond network analysis shows that the three channels are highly interconnected with similar energetics for hydronium as calculated for all paths near the OEC. The channels diverge as they approach the lumen, with the water chain in the broad channel better interconnected that in the narrow and large channels, where disruptions in the network are observed at about 10 [A] from the OEC. In addition, the barrier for hydronium translocation is lower in the broad channel, suggesting that a proton from the OEC could access the paths near the OEC, and likely exit to the lumen via the broad channel, passing through PsbO.

Ferredoxin-plastoquinone reductase (FQR) activity during cyclic electron flow (CEF) was first ascribed to the cytochrome b6f complex (b6f). However, this was later dismissed since b6f inhibition by antimycin-A (AA) could not be reproduced. AA presumably fails to ligate with haem bh, at variance with cytochrome bc1 complex, owing to a specific Qi-site occupation in b6f. Currently, PROTON GRADIENT REGULATION5 (PGR5) and the associated PGR5-Like1 are considered as FQR in the AA-sensitive CEF pathway. Here, we show that the b6f is conditionally inhibited by AA in a PGR5-independent manner when CEF is promoted. AA inhibition, demonstrated by single b6f turnover and electron transfer measurements, coincided with an altered Qi-site function which required Stt7 kinase activation by a strongly reduced plastoquinone pool. Thus, PGR5 and Stt7 were necessary for b6f activity and AA-sensitive electron transfer in CEF-favouring conditions. Extending previous findings, a new FQR activity model of the b6f is discussed.

Thiol-dependent redox regulations of enzyme activities play a central role in regulating photosynthesis. Beside the regulation of metabolic pathways, alternative electron transport has been shown to be subjected to thiol-dependent regulation. We investigated the regulation of O2 reduction at photosystem I. The level of O2 reduction in leaves and isolated thylakoid membranes depends on the photoperiod in which plants are grown. We used a set of Arabidopsis mutant plants affected in the stromal, membrane and lumenal thiol network to study the redox protein partners involved in regulating O2 reduction. Light-dependent O2 reduction was determined in leaves and in thylakoids of plants grown in short day and long day conditions using a spin-trapping EPR assay. In wild type samples from short day, ROS generation was twice the amount of that in samples from long day, while this difference was abolished in several redoxin mutants. An in vitro reconstitution assays showed that thioredoxin m, NADPH-dependent reductase C (NTRC) and NADPH are required for high O2 reduction levels in long day thylakoids. Using isolated photosystem I, we also show that reduction of a PSI protein is responsible for the increase in O2 reduction. Furthermore, differences in the membrane localization of thioredoxins m and 2-Cys peroxiredoxin were demonstrated between thylakoids of short day and long day plants. Finally, we propose a model of redox regulation of O2 reduction according to the reduction power of the stroma and the capabilities of the different thiol-containing proteins to form a network of redox interactions.

Light-dependent protochlorophyllide oxidoreductase (LPOR) has captivated the interest of the research community for decades. One reason is the photocatalytic nature of the reaction catalyzed by the enzyme, and the other is the involvement of LPOR in the formation of a paracrystalline lattice called a prolamellar body (PLB) that disintegrates upon illumination, initiating a process of photosynthetic membrane formation. In this paper, we have integrated three traditional methods previously employed to study the properties of the enzyme to investigate how LPOR evolved and how PLB forms. We found that in cyanobacteria, LPOR activity appears to be independent of lipids, with membrane interaction primarily affecting the enzyme post-reaction, with MGDG and PG having opposite effects on SynPOR. In contrast, plant isoforms exhibit sequence alterations, rendering the enzyme effective in substrate binding mainly in the presence of anionic lipids, depending on residues at positions 122, 312, and 318. Moreover, we demonstrated that the interaction with MGDG could initially serve as enhancement of the substrate specificity towards monovinyl-protochlorophyllide (Pchlide). We have shown that the second LPOR isoforms of eudicots and monocots accumulated mutations that made these variants less and more dependent on anionic lipids, respectively. Finally, we have shown that in the presence of Pchlide, NADP+, and the lipids, plant but not cyanobacterial LPOR homolog remodel membranes into the cubic phase. The cubic phase is preserved if samples supplemented with NADP+ are enriched with NADPH. The results are discussed in the evolutionary context, and the model of PLB formation is presented. SignificanceLPOR is a unique enzyme with photocatalytic properties, developed by cyanobacteria and inherited by algae and plants. In this study, we investigated the properties of the cyanobacterial homolog, revealing that two lipids, PG and MGDG, have opposite effects on enzyme activity. Additionally, we identified mutations in plant isoforms that render the enzyme dependent on anionic lipids. Moreover, we demonstrated that in the presence of NADP+, the plant homolog remodels lipids into a cubic phase, which appears to be the initial step of prolamellar body (PLB) formation. PLB is a unique paracrystalline arrangement of lipids and proteins found in immature chloroplasts, which disintegrates upon illumination, initiating photosynthetic membrane formation.

AbstractHuman NQO1 is a flavoenzyme essential for the redox metabolization of many substances and associated with wide-impacting diseases such as cancer and Alzheime[r]s. Recent X-ray crystallographic studies have proposed that a few residues at the active site of NQO1 (including Tyr126 and Tyr128) may control enzyme catalysis and functional negative cooperativity. In this work, we use rapid mixing pre-steady state kinetics and hydrogen-deuterium exchange followed by mass spectrometry (HDX-MS) to evaluate experimentally the role of Tyr126 and Tyr128 in NQO1 functionality by generating mutants to Phe, Ala and Glu. Mutations to Phe caused mild effects, whereas those to Ala significantly decreased hydride transfer efficiency and those to Glu virtually abolished NQO1 activity. Interestingly, structural stability studies by HDX-MS showed significant perturbations particularly affecting the binding site of NADH/NAD+ in the less conservative mutations (particularly to Glu). Mutations of Tyr126 and Tyr128 seem to also modulate the non-synchronous catalysis in the two active sites (negative cooperativity) as well as the selectivity for NADH/NADPH as coenzymes. Our work experimentally demonstrates the critical role of Tyr126 and Tyr128 in the flavin reductive half-reaction of the catalytic cycle of NQO1 in the negative cooperativity, and also suggests that phosphorylation of these two Tyr residues might shut down NQO1 activity reversibly.

Carbonic anhydrase (CA) activity, associated with Photosystem II (PSII) from Pisum sativum, has been shown to enhance water oxidation. But, the nature of the CA activity, its origin and role in photochemistry has been under debate, since the rates of CA reactions, measured earlier, were less than the rates of photochemical reactions. Here, we demonstrate high CA activity in PSII from Pisum sativum, measured by HCO3- dehydration at pH 6.5 (i.e. under optimal condition for PSII photochemistry), with kinetic parameters Km of 2.7 mM; Vmax of 2.74{middle dot}10-2 mM{middle dot}sec-1; kcat of 1.16{middle dot}103 sec-1 and kcat/Km of 4.1{middle dot}105 M-1 sec-1, showing the enzymatic nature of this activity, which kcat exceeds by [~]13 times the rate of PSII, as measured by O2 evolution. The similar dependence of HCO3- dehydration, of the maximal quantum yield of photochemical reactions and of O2 evolution on the ratio of chlorophyll/photochemical reaction center II demonstrate the interconnection of these processes on the electron donor side of PSII. Since the removal of protons is critical for fast water oxidation, and since HCO3- dehydration consumes a proton, we suggest that CA activity, catalyzing very fast removal of protons, supports efficient water oxidation in PSII and, thus, photosynthesis in general.

A hypothetical protein encoded by Synechocystis sp. PCC 6803 open reading frame slr0201shows high sequence similarity to the C subunit of a group of unusual succinate dehydrogenases found in some archaeal species. Slr0201 was originally annotated as HdrB, the B subunit of heterodisulfide reductase, but appears to be SdhC instead. This protein was overexpressed in E. coli by cloning the PCR-derived slr0201 open reading frame into a pET16b-based expression vector. The overproduced Slr0201 accumulated predominantly in inclusion bodies with an apparent molecular mass of 33 kDa. The protein contained at least one [2Fe-2S] cluster based on UV-visible absorbance and CD spectra and EPR spectroscopy, in conjunction with stoichiometric analysis of protein-bound iron and sulfur content. Redox titration showed a midpoint potential (Em) of + 17 mV at pH 7.0, which is consistent with Slr0201 serving a role in transferring electrons between succinate and plastoquinone. Slr0201 was also overproduced in Synechocystis sp. PCC 6803 by introducing an additional, His-tagged slr0201 into the Synechocystis genome replacing psbA3, creating the slr0201+-His overexpression strain. Immunoblot analysis shows that Slr0201 is membrane-associated in the wild type. However, in the Slr0201+-His strain, immunoreaction occurred in both the membrane and soluble fractions, possibly as a consequence of processing near the N-terminus. The results obtained with Slr0201 are discussed in the light of one of the cyanobacterial SdhB subunits, which shares redox commonalities with archaeal SdhB.

Mitochondria play crucial roles as both the powerhouse and signaling center of cells, balancing cell survival and death to maintain homeostasis. Disruption of this balance can lead to various diseases. Therefore, exploring the components involved in mitochondrial metabolism presents a significant challenge. In this context, respiratory supercomplexes are evolutionarily conserved, stable associations between membrane complexes and molecules, including proteins and lipids, within the inner mitochondrial membrane. These supercomplexes dynamically respond to metabolic demands, enhancing the electron transfer rate and reducing the production of reactive oxygen species. Recent research has identified cytochrome c, a mobile electron carrier between complexes III and IV, as a potential key player in the formation of these supercomplexes. This study focuses on elucidating the role of cytochrome c in modulating the assembly of supercomplexes, using the yeast Saccharomyces cerevisiae as a model system for mitochondrial metabolism. Our findings indicate that the viability of Saccharomyces cerevisiae relies on the presence of cytochrome c, with both isoforms playing a role in the assembly of respiratory supercomplexes. Notably, isoform-2 of cytochrome c enhances electron transfer efficiency, resulting in reduced ROS production.

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in organello in real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U-13C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system inferred that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U-13C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

In enzymology, hysteresis is manifested as a time-dependent shift in the kinetic behavior of an enzyme. Through hysteresis, the activation or inhibition of a biological pathway can be regulated by a molecule or metabolite that acts as a hysteretic modulator of the enzyme within that metabolic route. This mechanism of regulation contrasts with those that act on gene expression leading to modulation of enzyme protein levels. Through hysteresis, the amplitude of natural oscillations in metabolic pathways can be adjusted according to the levels of a metabolite that might be beneficial for cells. At physiological level, the slow response of hysteretic enzymes to changes, in the cellular levels of substrates, allows a time-dependent buffering effect on certain metabolites. Understanding the mechanisms and properties of hysteretic enzymes has been important for developing new therapies and improving our understanding of these enzymes in biological systems. However, due to their complex kinetics, the study of hysteretic enzymes has remained a challenge over time. In this study, we characterized the reduction of cytochrome b5 by NADH-dependent microsomal enzymes from rat liver using recombinant purified cytochrome b5, coenzyme Q10 and coenzyme Q0, as substrates, to mimic the conditions found in biological membranes, where competition between cytochrome b5 and other substrates might influence their reduction. We found a lag-time-dependent behavior in the cytochrome b5 reduction compatible with the existence of hysteretic modulation induced by endogenous molecules present in these membranes. Our data suggest that at least for the case of coenzyme Q10, fluctuations in its levels may impact metabolic pathways in which reduced cytochrome b5 levels play a key for the function of the cytochrome b5-dependent route.

Inorganic polyphosphate, a linear polymer of orthophosphate residues linked by phosphoanhydride bonds, occurs in all three domains of life and plays a diverse and prominent role in metabolism and cellular regulation. While the polyphosphate metabolism and its physiological significance have been well studied in bacteria and eukaryotes including human, there are only few studies in archaea available so far. In Crenarchaeota including members of Sulfolobaceae, the presence of polyphosphate and degradation via exopolyphosphatase has been reported and there is some evidence for a functional role in metal ion chelation, biofilm formation, adhesion and motility, however, the nature of the crenarchaeal polyphosphate kinase is still unknown. Here we used the crenarchaeal model organism Sulfolobus acidocaldarius to study the enzymes involved in polyphosphate synthesis. The two genes annotated as thymidylate kinase (saci_2019 and saci_2020), localized downstream of the exopolyphosphatase, were identified as the missing polyphosphate kinase in S. acidocaldarius (SaPPK3). Thymidylate kinase activity was confirmed for Saci_0893. Notably Saci_2020 showed no polyphosphate kinase activity on its own but served as regulatory subunit (rPPK3) and was able to enhance polyphosphate kinase activity of the catalytically active subunit Saci_2019 (cPPK3). Heteromeric polyphosphate kinase activity is reversible and shows a clear preference for polyP-dependent nucleotide kinase activity, i.e. polyP-dependent formation of ATP from ADP (12.4 U/mg) and to a lower extent of GDP to GTP whereas AMP does not serve as substrate. PPK activity in the direction of ATP-dependent polyP synthesis is rather low (0.25 U/mg); GTP was not used as phosphoryl donor. A combined experimental modelling approach using quantitative 31P NMR allowed to follow the reversible enzyme reaction for both ATP and polyP synthesis. PolyP synthesis was only observed when the ATP/ADP ratio was kept high, using an ATP recycling system. In absence of such a recycling system, all incubations with polyP and PPK would reach an equilibrium state with an ATP/ADP ratio between 3 and 4, independent of the initial conditions. Structural and sequence comparisons as well as phylogenetic analysis reveal that the S. acidocaldarius PPK is a member of a new PPK family, named PPK3, within the thymidylate kinase family of the P-loop kinase superfamily, clearly separated from PPK2. Our studies show that polyP, in addition to its function as phosphate storage, has a special importance for the energy homeostasis of S. acidocaldarius and due to its reversibility serves as energy buffer under low energy charge enabling a quick response to changes in cellular demand.