Biochimica et Biophysica Acta (BBA) - Bioenergetics

Molecular hydrogen (H2) is produced by plants under hypoxia and has been implicated in stress acclimation, yet its enzymatic source in vascular plants remains unknown because canonical hydrogenases are absent from angiosperm genomes. Here, using thermodynamic modeling, kinetic analysis, pharmacological perturbation, substrate-feeding assays, metabolite profiling, and cross-species comparison, we identify a mitochondrial origin for hypoxic H2 production in plants and define its biochemical requirements. Our data reveal a hydrogenase-like activity that obligatorily couples to Complex I Fe-S/quinone branch turnover and exhibits the hallmark of flavin-based electron bifurcation, a mechanism that cannot be explained by simple flavin over-reduction. The rotenone paradox, in which blockade of the N2-to-ubiquinone step abolishes rather than enhances H2 production, constitutes the most diagnostic evidence for this mechanism. This activity is promoted by convergent NADH- and succinate-supplying pathways, requires protonmotive force and continued ubiquinone re-oxidation through alternative oxidase, and is favored under micro-oxic and acidic conditions. The biochemical properties of this activity are consistent with a model in which a plant-specific Complex I assembly intermediate, termed CI*, provides the catalytic platform through an FMN-centered, N1a-assisted electron-bifurcation mechanism. Attempts to reconstitute H2 production from isolated Complex I subcomplexes have not yielded activity, consistent with the models prediction that this function requires an intact mitochondrial membrane system. H2-producing activity localized predominantly to mitochondria and was conserved across phylogenetically diverse vascular plants. Together, these findings reveal a previously unrecognized electron-bifurcating activity associated with a plant Complex I assembly intermediate. They provide a mechanistic framework for understanding endogenous H2 emission under hypoxia, a phenomenon first reported over 60 years ago but never explained, and identify mitochondrial redox flexibility as a potential target for improving tolerance to flooding and other oxygen-limiting stresses.

Photoactivated intermolecular electron transfer (ET) in cryptochromes proceeds along chains of aromatic residues and creates a spatially separated pair of radical electrons. Ultrafast time-dependent spectroscopy can provide experimental insight into this process and theoretical estimates of charge transfer rates are commonly obtained via Marcus theory. Here, we present a new perspective on the ET in European robin cryptochrome 4a (ErCry4a) that synthesizes insights from real-time ET calculations, ultrafast spectroscopic measurements and analytical derivations. The simulations exemplify that molecular vibrations play an essential role in enabling the ET dynamics, which was further rationalized through analytical derivations. Ultrafast pump-probe spectroscopy provided experimental access to the first 1.5 ns of the ET cascade, where multiple radical pair recombination rates arise due to the dynamic equilibrium along the ET chain. We show that the motions of the protein environment and the ET dynamics are inseparably coupled, violating the timescale separation required for Marcus theory. The presented results highlight that non-equilibrium coupling between electronic and nuclear motion dominates ET kinetics in ErCry4a during the first nanosecond after photo-excitation. The findings exemplify the limits of Marcus theory and refine the interpretation of ultrafast spectroscopic signatures in cryptochromes.

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.

Cytochrome bc1 is one of the key enzymes of biological energy-conserving systems. In its catalytic Q cycle, the central reaction is the oxidation of quinol (QH2), upon which electrons are directed to two separate cofactor chains. The molecular mechanism of this reaction remains elusive. The canonical model, assuming a sequence of reactions dictated by the equilibrium redox midpoint potentials of cofactors (the 2Fe2S cluster and heme bL), has recently been challenged by a new model of EB derived from quantum mechanical (QM) calculations - EMET (EMergent Electron Transfer) (https://doi.org/10.1021/acsomega.5c13233). These two models predict fundamentally different microstates of the enzyme in which semiquinone (SQ) is formed in the catalytic site (Q o) and also predict different lowest-energy configurations. Here, we test these predictions using EPR spectroscopy on highly concentrated preparations of isolated bacterial cytochrome bc1. We detect SQ spin-coupled to the reduced 2Fe2S cluster (2Fe2Sred), whose population markedly exceeds that of reduced heme bL and forms exclusively in sites containing oxidized heme. We also identify that the lowest-energy configuration corresponds to the state with reduced heme bH (adjacent to heme bL), oxidized heme bL and SQ-2Fe2Sred. These two features are precluded by the canonical model but are consistent with EMET. We conclude that EMET, unlike the canonical EB model, satisfactorily describes the occurrence of stochastic, spin-selective processes that result in electron stoichiometry among hemes b, the 2Fe2S cluster, and SQ at Qo that are observed spectroscopically.

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.

The carotid body (CB) is the key peripheral oxygen sensor. CB mitochondria are hypothesised to be uniquely adapted with unusually low intrinsic oxygen affinity which, in association with nitric oxide (NO) and reactive oxygen species signalling, enables acute responsiveness to hypoxia. However, CB mitochondrial physiology or intrinsic oxygen affinity have never been measured directly. We sought to address this key gap by isolating sheep CB mitochondria and comprehensively characterising their phenotype and contrasting them to a non-oxygen sensing tissue, left ventricular myocardium (LV). High resolution respirometry, liquid chromatography mass spectrometry, enzymatic assays and in silico modelling were used to characterise mitochondrial content, aerobic capacity, oxygen affinity, complex subunit abundance and activity, H2O2 production and NO sensitivity in ovine CB and LV. Mitochondrial oxygen affinity (P50 = 0.089 mmHg) was lower in the CB than the LV (P50 = 0.058 mmHg; p = 0.005). Whilst mitochondrial content was lower in the CB, CB mitochondria had higher respiratory rates and enzymatic activity than LV. H2O2 production and NO sensitivity were similar in the two tissues. While intrinsic mitochondrial oxygen affinity is slightly lower in the oxygen sensing CB than in the non-oxygen sensing LV, this difference is small. Hence, any role of mitochondria in CB oxygen sensing is not due to an intrinsic difference in the O2 affinity of cytochrome oxidase due to differential expression of its subunits. Instead, this work suggests that differences in O2 affinity in vivo are secondary to other factors, perhaps including NO, that alter mitochondrial O2 affinity.

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

Whether Photosystem I (PSI) core subunits accumulate prior to light exposure in developing angiosperm seedlings remains unresolved, with conflicting reports across species. Here, we investigated the presence and membrane colocalization of the PSI core subunit PsaA in etioplasts of dark-grown angiosperms representing dicot and monocot species. Immunoblotting showed that PsaA accumulates in etioplasts of all three dicot species examined (pea, Arabidopsis, and runner bean), whereas in the monocot oat it was detected only after prolonged etiolation, at substantially lower levels and with an anomalously high apparent molecular weight. Blue-native PAGE analysis reveals that a fraction of PsaA co-migrates with LPOR, PsaB, FNR, and chlorophyll synthase, suggesting co-localization within a shared membrane microdomain rather than stable complex formation. The thylakoid insertase Alb3 was more abundant in dicot etioplasts, consistent with a potential role in the early integration of PsaA into the membrane. Upon illumination, pea reached PSI functionality faster than oat, with P700 oxidation detectable 30 min earlier, linking the dark accumulation of PsaA to an accelerated photosynthetic onset. These findings demonstrate light-independent accumulation of a PSI core subunit in a species-dependent manner and point to early steps in PSI biogenesis that precede full photosynthetic complex assembly. Highlight Contrary to prevailing models, a Photosystem I core subunit PsaA accumulates in dark-grown angiosperm seedlings before light exposure, revealing light-independent early steps in photosynthetic complex biogenesis.

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

Gills are multifunctional organs that integrate respiration with homeostasis, including energy demanding processes such as osmoregulation and excretion. In osmoregulating decapod crustaceans, two spatially segregated gill types differ in function, ultrastructure and membrane composition, as well as in their responses to environmental change. Yet mitochondrial function in crab gills remains poorly characterized. Here, for the first time, we used high-resolution respirometry with a substrate-uncoupler-inhibitor titration (SUIT) protocol to characterize the mitochondrial phenotypes in anterior (respiratory) and posterior (osmoregulatory) gills. For this, gill filaments of the shore crab Carcinus maenas were permeabilized for 30 min in a saponin solution (optimized for each tissue at 25 {micro}g or 5 {micro}g saponin {middle dot} mg-1 gill fresh weight for anterior and posterior gills, respectively). Anterior gills exhibited higher leak control ratios (L/P, L/E), consistent with a leak-dominated mitochondrial phenotype that may contribute to redox balance at expense of maximal ATP yield. In contrast, posterior gills, displayed a higher phosphorylation control ratio and tighter coupling (higher Net P), reflecting a tightly-coupled, ATP-producing mitochondrial phenotype, in line with their role in sustaining ATP-intensive activities such as osmoregulation and excretion. Our results revealed that anterior and posterior gills operate as "two engines in one organ": by quantifying how each gill type partitions respiratory capacity between phosphorylation and leak pathways, this study provides a mechanistic framework for understanding how mitochondrial specialization supports functional division of labour within a single organ and contributes to physiological adaptation to dynamically fluctuating marine environments.

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

Human glutathione synthetase (hGS) is a negatively cooperative ATP-grasp enzyme that catalyzes the final step in the biosynthesis of glutathione, a tripeptide antioxidant critical for life. hGS functions as an obligate homodimer with one active site per subunit; the two active sites are separated by [~]40 Angstroms. How ligand binding in one subunit reshapes the distant partner active site has remained a central unresolved question in understanding hGS regulation. This study provides the first atomistic model of ligand-dependent inter-subunit communication underlying negative cooperativity in hGS. Using atomistic simulations and dynamical network analysis, this study reveals how reactant- and product-bound states remodel the empty partner active site, redistribute inter-subunit interactions, and organize long-range communication between the two active sites. The product-bound/partner-empty state displayed a larger and less hydrated empty active site, demonstrating that ligand identity in one subunit alters both the geometry and solvent environment of the opposite site. Changes in ligand-dependent interactions are distributed across the dimer interface, with prominent contributions from the 42-46 interface region, the 11-30 region, and the 212-236 helical/interface region. Suboptimal path analysis shows product- and reactant-bound states share a communication scaffold, with 64.1% of transmission residues common to both pathways, 30.8% product-specific, and 5.1% reactant-specific. Together, the present results establish a detailed structural framework for hGS negative cooperativity in which ligand binding remodels a distributed allosteric network linking substrate-binding loops, the dimer interface, and the partner active site. More broadly, this work demonstrates how atomistic simulations can resolve long-range active-site coupling in multimeric enzymes and provides a foundation for experimental tests of allosteric transmission in hGS.

Lanthanides function as enzyme cofactors in bacteria, where they are widely distributed in pyrroloquinoline quinone-dependent 8-bladed beta-propeller dehydrogenases. No lanthanide-dependent enzymes, however, have been described outside prokaryotes. Here, we combined structural bioinformatics, phylogenetics, AlphaFold3 co-folding, coordination-sphere comparison, and quantum-mechanical cluster modeling to search for and rank putative lanthanide-coordinating 8-bladed beta-propeller enzymes in Eukarya. We identified candidate lanthanide-coordinating proteins in a diverse range of eukaryotes, predominantly plants and fungi, including species of clear industrial and agricultural relevance. A high-confidence subset matched validated bacterial Ln-binders based on both geometric similarity to canonical Ln-binding sites and on predicted Ln3+ versus Ca2+ selectivity. Our findings indicate that lanthanide biology likely extends beyond bacteria, with implications for plant, fungal, and broader eukaryotic metabolism, and warrant targeted biochemical investigation.

Several metabolites within the reductive tricarboxylic acid (rTCA) cycle have been found to form prebiotically. However, how these metabolites connect to each other and form rTCA cycle remains unresolved. The rTCA cycle is an ancient route and is considered significant for the emergence of life, since it connects to the routes of amino acids and nucleobases synthesis. A major challenge to complete the rTCA cycle under prebiotic conditions is the thermodynamically unfavorable reductive carboxylation of succinate to -ketoglutarate. Here, we address this challenge by using the nature of energy: nonequilibrium conditions. By calculating the changes in free energy, {Delta}G, of succinate to -ketoglutarate, and its downstream reactions: -ketoglutarate to glutamate and -ketoglutarate to isocitrate under different nonequilibrium conditions, we find that these two-step reactions are exergonic under nonequilibrium conditions at a 10000:1 reactant-to-product ratio at 1.013 bar, pH 10 and 70{degrees}C. To prove the concept, we catalyze succinate to glutamate at a 10000:1 reactant-to-product ratio, with NH2OH and sodium dithionite. The process is catalyzed by Fe(0), Fe3O4, and artificial proto-[4Fe4S] clusters in 1M NaCl at pH 10 and 70{degrees}C under 1 atm of 13CO2 for 48 hours. This nonequilibrium condition and one-pot system successfully promote the formation of -ketoglutarate through carbon fixation with succinate and its subsequent conversion to glutamate. These findings demonstrate nonequilibrium states enable -ketoglutarate formation through succinate and CO2, and suggest that a tendency toward natural thermodynamics may serve as a driving force for autocatalysis in the origin of life. ImportanceHow life began remains open, metabolism provides a key framework for origins. We use a simple and robust energetic principle to show that non-equilibrium conditions can drive the highly endergonic carboxylation step of the reverse tricarboxylic acid (rTCA) cycle, enabling one-pot synthesis of glutamate. This is work bridges the gap between protometabolites and protometabolsim, suggesting that metabolites may have accumulated first, creating concentration gradients that drove reactions and ultimately enabled the emergence of protometabolism. These findings provide a plausible pathway from prebiotic chemistry to the emergence of metabolism.

Glutathione peroxidase 4 (GPx4) is the primary enzyme reducing lipid hydroperoxides, preventing membrane oxidative damage and protecting against ferroptosis. GPx4 is known to engage with lipid headgroups through electrostatic interactions, positioning the substrate for reduction. This work reveals and characterizes binding of highly anionic phosphoinositides (PIP lipids) by GPx4. PIPs are vital lipids in human cells and are central to many signaling processes, particularly in cytosolic facing membranes. Lipid overlay assays confirm interactions between GPx4 and phosphorylated PIPs, comparable to known anionic lipid binders. Protein NMR describes the interaction between GPx4 and PIPs within micelles. The greatest resonance shifting occurs with trisphosphorylated PIP, suggesting that higher anionic charge leads to greater binding, a known driver of GPx4 substrate recognition. Preferred anionic interactions were also confirmed with titration and crystallographic structure analysis of inositol phosphate 4 (IP4). A headgroup-binding site on GPx4 is revealed to be proximal to the cationic membrane interaction site. In conjunction with molecular simulations, these results show that PIP lipid interactions allow full engagement of GPx4 with the membrane and positions the headgroup to allow the lipid tail to interact with the catalytic site. Understanding whether GPx4 preferentially interacts with PIPs will allow better understanding of the protective function of this essential enzyme and a mechanism that may protect essential lipid signaling pathways from oxidative damage. SignificanceThis study allows a deeper structural and mechanistic understanding of GPx4, the primary enzyme that reduces lipid hydroperoxides and prevents ferroptosis. Gaining an understanding of phosphoinositide binding to GPx4 reveals a mechanism for potential preservation of these important signaling molecules and for ferroptosis protection. Observation of a specific binding site for headgroup engagement reveals a plausible lipid interaction mode and functional mechanism of this important cytoprotective enzyme.

Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H2 uptake, HoxEFUYH utilizes NAD+ and oxidized ferredoxin, whereas H2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from dark carbohydrate oxidation. This requires reassessment of current strategies for green H2 production in cyanobacteria.

Malonate is often described as an endogenous inhibitor of complex II of the electron transport chain. However, the cellular source of malonate is unclear, and current knowledge concerning its metabolism is limited to the action of a single enzyme, Acyl-CoA Synthetase Family Member 3 (ACSF3), which converts malonate to malonyl-CoA in the mitochondrial matrix. One potential route of malonate metabolism downstream of ACSF3 is its consumption by the mitochondrial fatty acid synthesis (mtFAS) pathway. However, studies examining the link between ACSF3 and mtFAS have yielded conflicting results. We developed a novel mass spectrometry approach to perform stable isotope tracing into products of mtFAS, and found that while malonate is in fact a carbon source for mtFAS, ACSF3 is not required for malonate incorporation into mtFAS products. Using this method to trace other nutrients into mtFAS, we also found evidence of acetyl-CoA carboxylase 1 (ACC1)-dependent malonate synthesis from glucose. We further show that ACC1 is required for optimal mtFAS activity, with downstream effects on oxidative phosphorylation. Together these findings establish the malonate as a regulated endogenous intermediate that supports mtFAS activity and mitochondrial oxidative function.

Biosynthesis of the linear tetrapyrrole phycocyanobilin (PCB) by the ferredoxin-dependent bilin reductase (FDBRs) PcyA is essential for light-harvesting and regulatory processes in diverse photosynthetic organisms, yet its evolutionary origins are not fully understood. PcyA evolved from pre-PcyA proteins found in diverse bacteria. Three lineages of pre-PcyA proteins were identified: Pre-1, Pre-2 and Pre-3. Using an in vivo co-expression assay, Pre-2 and Pre-3 proteins were shown to be active FDBRs that did not synthesize PCB, whereas Pre-1 activity was apparently low. In refining these results, we noted a discrepancy between phycoerythrobilin populations generated by Pre-3 and by the distantly related FDBR PebS. We therefore examined the properties of pre-PcyA enzymes in vitro, using an updated pre-PcyA phylogeny to select an alternative pre-1 target. Biochemical analyses revealed that Pre-1 and Pre-2 catalyze the two-electron reduction of biliverdin (BV) to 3E-phytochromobilin (3E-P[FE]B), in contrast to the known synthesis of 3Z-phytobilins by other FDBRs. Pre-3 can also carry out an additional two-electron reduction to yield 3E-phycoerythrobilin (3E-PEB), again distinct from the 3Z-PEB produced by PebS. We then used comparative sequence and structure analysis to target candidate catalytic residues for site-directed mutagenesis. Variant Pre-1 exhibited altered product stereochemistry, but no effects on Pre-2 were observed and Pre-3 variants unexpectedly gained the ability to bind cyclic tetrapyrroles. These findings underscore the plasticity and promiscuity of this enzyme family. Together, this work illustrates how the flexible catalytic potential of ancestral enzymes shaped the evolution and diversification of bilin biosynthetic pathways.

Photosynthetic membranes undergo structural remodeling in response to environmental stress by altering fatty acid composition and desaturation levels. These changes, mediated by fatty acid desaturases (FADs), are essential for maintaining photosynthetic performance and adaptation. In this study, we demonstrate that copper-deficient Chlamydomonas reinhardtii cells upregulate the expression of the gene encoding stearoyl-ACP desaturase (SAD/FAB2). We propose that this four-fold induction reflects an increased physiological demand for its primary product, oleic acid (18:1{Delta}9), and its subsequent downstream derivatives. The sad mutants exhibit a significant reduction in 18:1{Delta}9 content compared to wild-type cells, which correlates with diminished growth rates. Although SAD abundance increases under Cu deficiency, loss of SAD strongly alters C18 fatty acid composition across Cu conditions, while the growth defect is most apparent under Cu-replete conditions. This suggests that SAD activity may be a limiting factor in copper-depleted environments, leading to slower growth and reduced 18:1{Delta}9 levels in the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both of which are critical for photosynthetic function. The desaturation reaction catalyzed by SAD requires molecular oxygen and electrons supplied by ferredoxin (Fd). Using reciprocal IP-MS, we identified FDX5 as a Cu-deficiency specific SAD interacting protein. However, fdx5 mutants retained wild-type fatty acid profiles, indicating that FDX5 is not strictly required for SAD-dependent lipid desaturation and that another ferredoxin, likely FDX1, can compensate.

Mycobacterium tuberculosis fatty acid synthase I (Mtb FAS-I) is a multifunctional hexameric complex essential for fatty acid (FA) synthesis. The need of a hexameric structure for activity of the complex in Mtb remains elusive. Here, we model a conformation of the functionally active complex with acyl carrier protein (ACP) at ketoacyl synthase (KS). Our model reveals a crucial cross-dome dependence in the mechanism of FA synthesis at the condensation step. Using molecular dynamics simulation, we identify key ACP and KS residues that mantain persistent interactions. ACPs phosphopantetheine (PPT) arm adopts several conformations while accessing KSs catalytic pocket, including two distinct conformations that correlate with volumes of ACP and KS pockets. A PHE residue, reported as a gatekeeper of the KS pocket in other species, also shows open and closed orientations in our simulation. Our results provide crucial insights that are essential for a mechanistic undersanding of the Mtb FAS-I complex.