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Abstract Monotopic membrane-bound flavoproteins, sulfide:quinone oxidoreductases (SQRs), have a variety of physiological functions, including sulfide detoxification. SQR enzymes are classified into six groups. SQRs use the flavin adenine dinucleotide (FAD) cofactor to transfer electrons from sulfide to quinone. A type VI SQR of the photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina (TrSqrF), has been previously characterized, and the mechanism of sulfide oxidation has been proposed. This paper reports the characterization of quinone binding site (QBS) of TrSqrF composed of conserved aromatic and apolar amino acids. Val331, Ile333, and Phe366 were identified near the benzoquinone ring of enzyme-bound decylubiquinone (dUQ) using the TrSqrF homology model. In silico analysis revealed that Val331 and Ile333 alternately connected with the quinone head group via hydrogen bonds, and Phe366 and Trp369 bound the quinones via hydrophobic interactions. TrSqrF variants containing alanine (V331A, I333A, F366A) and aromatic amino acid (V331F, I333F, F366Y), as well as a C-terminal α-helix deletion (CTD) mutant were generated. These amino acids are critical for quinone binding and, thus, catalysis. Spectroscopic analyses proved that all mutants contained FAD. I333F replacement resulted in the lack of the charge transfer complex. In summary, the interactions described above maintain the quinone molecule’s head in an optimal position for direct electron transfer from FAD. Surprisingly, the CTD mutant retained a relatively high level of specific activity while remaining membrane-anchored. This is a unique study because it focuses on the QBS and the oxidative stage of a type VI sulfide-dependent quinone reduction. Key points • V331, I333, F366, and W369 were shown to interact with decylubiquinone in T. roseopersicina SqrF • These amino acids are involved in proper positioning of quinones next to FAD • I333 is essential in formation of a charge transfer complex from FAD to quinone Graphical abstract

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

The medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]-spiroketal pharmacophore that was recently shown to be assembled by flavin-dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]-spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon-carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals., Angewandte Chemie. International Edition, 60 (52), ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain’s sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury., The brain is sensitive to oxygen deprivation. Here, the authors show in experimental animals that sensitivity to hypoxia is inversely related to the level of sulfide:quinone oxidoreductast (SQOR) and the capacity to catabolize sulfide in the brain.

A phytoalexin, Resveratrol remains a legendary anticancer drug candidate in the archives of scientific literature. Although earlier wet-lab experiments rendering its multiple biological targets, for example, epidermal growth factors, Pro-apoptotic protein p53, sirtuins, and first apoptosis signal (Fas) receptor, Mouse double minute 2 (MDM2) ubiquitin-protein ligase, Estrogen receptor, Quinone reductase, etc. However, notwithstanding some notable successes, identification of an appropriate Resveratrol target(s) has remained a major challenge using physical methods, and hereby limiting its translation into an effective therapeutic(s). Thus, computational insights are much needed to establish proof-of-concept towards potential Resveratrol target(s) with minimum error rate, narrow down the search space, and to assess a more accurate Resveratrol signaling pathway/mechanism at the starting point. Herein, a brute-force technique combining computational receptor-, ligand-based virtual screening, and classification-based machine learning, reveals the precise mechanism of Resveratrol action. Overall, MDM2 ubiquitin-protein ligase (4OGN.pdb) and co-crystallized quinone reductases 2 (4QOH.pdb) were found two suitable drug targets in the case of Resveratrol derivatives. Indeed, carotenoid cleaving oxygenase together with later twos gave gigantic momentum in guiding the rational drug design of Resveratrol derivatives. These molecular modeling insights would be useful for Resveratrol lead optimization into a more precise science.Communicated by Ramaswamy H. Sarma.

To ensure the physical interaction between a protein and its ligand, many techniques can be applied. One of them, isothermal titration calorimetry (ITC), measures the heat exchange between a forming molecular complex and its milieu. From this heat exchange, it is possible to acquire the thermodynamic parameters, the binding stoichiometry and the affinity constant (K

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein.

Melatonin, (N-acetyl-5-methoxytryptamine), is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule. For a long time, melatonin had been described as a ligand of a putative "receptor" present in mammalian brains named MT

The third melatonin binding site MT

Long non-coding RNAs (lncRNAs) play critical roles in human cancers. HOXA11 anti-sense RNA (

Consumption of Cassia occidentalis (CO) seeds, a ubiquitously distributed weed plant, is responsible for a pathological condition known as hepato-myo-encephalopathy (HME). The toxicity of CO seeds is largely attributed to the presence of anthraquinones (AQs). Here, we report that Emodin, a CO anthraquinone, inhibits the enzymatic activity of NADPH-Quinone reductase, which is an intracellular enzyme fundamentally involved in the detoxification of quinone containing compounds. Emodin binds to the active site of the enzyme and acts as a competitive inhibitor with respect to 2, 6-Dichlorophenolindophenol, a known substrate of NADPH-Quinone reductase. Moreover, our in-vitro study further revealed that Emodin was cytotoxic to primary rat hepatocytes.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

The pyrroloquinoline quinone (PQQ)-dependent dehydrogenase DepA detoxifies deoxynivalenol (DON) by converting the C3-OH into a keto group. Herein, two crystal structures of DepA and its complex with PQQ were determined, together with biochemical evidence confirming the interactions of DepA with PQQ and DON and revealing a unique tyrosine residue important for substrate selection. Furthermore, four loops over the active site essential for DepA activity were identified, of which three loops were stabilized by PQQ, and the fourth loop invisible in both structures was considered important for binding DON, together constituting a lid for the active site. Preliminary engineering of the loop showed its potential for enzyme improvement. This study provides structural insights into how a PQQ-dependent dehydrogenase is equipped with the function of DON conversion and for the first time shows the necessity of a lid structure for PQQ-dependent dehydrogenase activity, laying foundation for structure-based design to enhance catalysis efficiency.

The Na

The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

Sulfur-oxidizing bacteria can oxidize hydrogen sulfide (H(2)S) to produce sulfur globules. Although the process is common, the pathway is unclear. In recombinant Escherichia coli and wild-type Corynebacterium vitaeruminis DSM 20294 with sulfide:quinone oxidoreductase (SQR) but no enzymes to oxidize zero valence sulfur, SQR oxidized H(2)S into short-chain inorganic polysulfide (H(2)S(n), n ≥ 2) and organic polysulfide (RS(n)H, n ≥ 2), which reacted with each other to form long-chain GS(n)H (n ≥ 2) and H(2)S(n) before producing octasulfur (S(8)), the main component of elemental sulfur. GS(n)H also reacted with glutathione (GSH) to form GS(n)G (n ≥ 2) and H(2)S; H(2)S was again oxidized by SQR. After GSH was depleted, SQR simply oxidized H(2)S to H(2)S(n), which spontaneously generated S(8). S(8) aggregated into sulfur globules in the cytoplasm. The results highlight the process of sulfide oxidation to S(8) globules in the bacterial cytoplasm and demonstrate the potential of using heterotrophic bacteria with SQR to convert toxic H(2)S into relatively benign S(8) globules. IMPORTANCE Our results provide evidence of H(2)S oxidation producing octasulfur globules via sulfide:quinone oxidoreductase (SQR) catalysis and spontaneous reactions in the bacterial cytoplasm. Since the process is an important event in geochemical cycling, a better understanding facilitates further studies and provides theoretical support for using heterotrophic bacteria with SQR to oxidize toxic H(2)S into sulfur globules for recovery.

The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10–100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.

Mitochondrial dysfunction is related to common age-related disorders, including neurodegenerative diseases, metabolic syndrome, and carcinogenesis. Therefore, maintaining the functionality and integrity of mitochondria is important for human health. Herein, we found that sulfide:quinone oxidoreductase (Sqr), which oxidizes hydrogen sulfide to reactive sulfur species (RSS), was indispensable to mitochondria health in the eukaryotic model microorganism Schizosaccharomyces pombe. Sqr knock-out led to morphological changes and functional deficiencies of mitochondria and apoptosis in S. pombe. The Sqr knock-out strain displayed the same phenotypes as the cysteine-synthesis-deficient strain, and cysteine addition complemented the effects caused by Sqr knock-out. In S. pombe, Sqr was the main RSS producer in mitochondria, and RSS instead of H2S was used by cysteine synthase to synthesize cysteine. This finding rewrites the cysteine biosynthesis route in S. pombe and may also in other eukaryotes and prokaryotes, and highlights the importance of cysteine and RSS in maintaining mitochondrial health., Highlights • Sqr is an important RSS producer in mitochondria. • RSS is involved in cysteine de novo biosynthesis. It is the de facto substrate of cysteine synthase. • Sqr is required for maintaining the health of mitochondria, might be a new target for inhibiting cell proliferation.

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100-400 s-1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.

Unlike most other tissues, the colon epithelium is exposed to high levels of H(2)S derived from gut microbial metabolism. H(2)S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H(2)S exposure as they harbor an efficient mitochondrial H(2)S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host–microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H(2)S poisoning. H(2)S at concentrations ≤20 μm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H(2)S concentrations as low as 5 μm poisoned cells. Mitochondrial H(2)S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD(+)/NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H(2)S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H(2)S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H(2)S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming

PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO), but no evidence at the transcript or protein level has been presented. In this study, PA0660 was purified and its biochemical and kinetic properties were characterized. Absorption spectroscopy and mass spectrometry demonstrated a tightly, noncovalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ∼30-fold larger kcat/ Km and kred/ Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lesser extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with a lack of azoreductase activity. The enzyme turned over through a ping-pong bi-bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple-sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1000 open reading frames currently annotated as hypothetical NMOs. Our results suggest that PA0660 should be classified as an NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Two new physalins, 7α-hydroxy-5-deoxy-4-dehydrophysalin IX (

Our previous work showed that many lichenized Ascomycetes can generate hydroxyl radicals using quinone-based extracellular redox cycling. During cycling, hydroquinones must be formed and subsequently regenerated from quinones using a quinone reductase (QR). However, we also showed that no simple correlation exists between QR activity and rates of hydroxyl radical formation. To further investigate the role of QR in hydroxyl radical formation, three model lichen species, Leptogium furfuraceum, Lasallia pustulata and Peltigera membranacea were selected for further investigation. All possessed QR activity and could metabolize quinones, and both Leptogium furfuraceum and Lasallia pustulata actively produced hydroxyl radicals. By contrast, P. membranacea produced almost no hydroxyl radicals, and although the lichen readily metabolized quinones, no hydroquinone production was detected. Peltigera had laccase (LAC) activity that was c. 50 times higher than in the other two species, suggesting that LAC rapidly oxidizes the hydroquinones, preventing radical formation deriving from auto-oxidation. It appears that in some lichens hydroxyl radical formation is blocked by the presence of high redox enzyme activity. QR from P. didactyla was studied further and found to display similar properties to the enzyme from free-living fungi, although it possessed an unusually high molecular mass (c. 62 kDa).

NAD(P)H: Quinone Oxidoreductase 1 (NQO1) is an antioxidant enzyme that catalyzes the two-electron reduction of several different classes of quinone-like compounds (quinones, quinone imines, nitroaromatics, and azo dyes). One-electron reduction of quinone or quinone-like metabolites is considered to generate semiquinones to initiate redox cycling that is responsible for the generation of reactive oxygen species and oxidative stress and may contribute to the initiation of adverse drug reactions and adverse health effects. On the other hand, the two-electron reduction of quinoid compounds appears important for drug activation (bioreductive activation) via chemical rearrangement or autoxidation. Two-electron reduction decreases quinone levels and opportunities for the generation of reactive species that can deplete intracellular thiol pools. Also, studies have shown that induction or depletion (knockout) of NQO1 were associated with decreased or increased susceptibilities to oxidative stress, respectively. Moreover, another member of the quinone reductase family, NRH: Quinone Oxidoreductase 2 (NQO2), has a significant functional and structural similarity with NQO1. The activity of both antioxidant enzymes, NQO1 and NQO2, becomes critically important when other detoxification pathways are exhausted. Therefore, this article summarizes the interactions of NQO1 and NQO2 with different pharmacological agents, endogenous biochemicals, and environmental contaminants that would be useful in the development of therapeutic approaches to reduce the adverse drug reactions as well as protection against quinone-induced oxidative damage. Also, future directions and areas of further study for NQO1 and NQO2 are discussed.

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°′ ∼−450 mV) using NADH (E°′ −320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°′ −80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

A multitude of effects has been attributed to melatonin at pmol/L to mmol/L concentrations. More than fifteen targets have been proposed for melatonin but only few of them are well characterized. The current guidelines intend to provide a framework to improve and rationalize the characterization of melatonin targets and effects. They should be considered as mandatory guidelines and minimum requirements for manuscripts submitted to the Journal of Pineal Research.

International audience; Hydrogen sulfide, a signaling molecule formed mainly from cysteine, is catabolized by sulfide:quinone oxidoreductase (geneSQOR). Toxic hydrogen sulfide exposure inhibits complex IV. We describe children of two families with pathogenic variants inSQOR. Exome sequencing identified variants; SQOR enzyme activity was measured spectrophotometrically, protein levels evaluated by western blotting, and mitochondrial function was assayed. In family A, following a brief illness, a 4-year-old girl presented comatose with lactic acidosis and multiorgan failure. After stabilization, she remained comatose, hypotonic, had neurostorming episodes, elevated lactate, and Leigh-like lesions on brain imaging. She died shortly after. Her 8-year-old sister presented with a rapidly fatal episode of coma with lactic acidosis, and lesions in the basal ganglia and left cortex. Muscle and liver tissue had isolated decreased complex IV activity, but normal complex IV protein levels and complex formation. Both patients were homozygous for c.637G > A, which we identified as a founder mutation in the Lehrerleut Hutterite with a carrier frequency of 1 in 13. The resulting p.Glu213Lys change disrupts hydrogen bonding with neighboring residues, resulting in severely reduced SQOR protein and enzyme activity, whereas sulfide generating enzyme levels were unchanged. In family B, a boy had episodes of encephalopathy and basal ganglia lesions. He was homozygous for c.446delT and had severely reduced fibroblast SQOR enzyme activity and protein levels. SQOR dysfunction can result in hydrogen sulfide accumulation, which, consistent with its known toxicity, inhibits complex IV resulting in energy failure. In conclusion, SQOR deficiency represents a new, potentially treatable, cause of Leigh disease.

Photochemoprotection of the skin can be achieved by inhibiting inflammation and oxidative stress, which we tested using Cordia verbenacea extract, a medicinal plant known for its rich content of antioxidant molecules and anti-inflammatory activity. In vitro antioxidant evaluation of Cordia verbenacea leaves ethanolic extract (CVE) presented the following results: ferric reducing antioxidant power (886.32 μM equivalent of Trolox/g extract); IC50 of 19.128 μg/ml for scavenging 2,2-diphenyl-1-picrylhydrazyl; IC50 of 12.48 μg/mL for scavenging 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); decrease of hydroperoxides from linoleic acid (IC50 of 10.20 μg/mL); inhibition of thiobarbituric acid reactive substances (IC50 8.90 μg/mL); iron-chelating ability in bathophenanthroline iron assay (IC50 47.35 μg/mL); chemiluminescence triggered by free radicals in the H2O2/horseradish peroxidase/luminol (IC50 0.286 μg/mL) and xanthine/xanthine oxidase/luminol (IC50 0.42 μg/mL) methods. CVE (10–100 mg per kg, 30 min before and immediately after UVB exposure) treatment was performed by gavage in hairless mice. CVE inhibited skin edema, neutrophil infiltration, and overproduction of MMP-9; reduced levels of TNF-α, IL-1β, and IL- 6; numbers of skin mast cells, epidermal thickening, number of epidermal apoptotic keratinocytes, and collagen degradation. CVE increased the skin's natural antioxidant defenses as observed by Nrf-2, NAD(P)H quinone oxidoreductase 1, and heme oxygenase 1 mRNA expression enhancement. Furthermore, CVE inhibited lipid peroxidation and superoxide anion production and recovered antioxidant reduced glutathione, catalase activity, and ROS scavenging capacity of the skin. Concluding, CVE downregulates the skin inflammatory and oxidative damages triggered by UVB, demonstrating its potentialities as a therapeutic approach.

Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redox-active centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class.

N-ribosyldihydronicotinamide:quinone oxidoreductase 2 (NQO2/QR2, Enzyme Commission number 1.10.99.2) is a cytosolic enzyme, abundant in the liver and variably expressed in mammalian tissues. Cloned 30 years ago, it was characterized as a flavoenzyme catalyzing the reduction of quinones and pseudoquinones. To do so, it uses exclusively N-alkyl nicotinamide derivatives, without being able to recognize NADH, the reference hydrure donor compound, in contrast to its next of a kind, NAD(P)H:quinone oxidoreductase 1 (NQO1). For a long time both enzymes have been considered as key detoxifying enzymes in quinone metabolism, but more recent findings point to a more toxifying function of NQO2, particularly with respect to ortho-quinones. In fact, during the reduction of substrates, NQO2 generates fairly unstable intermediates that reoxidize immediately back to the original quinone, creating a futile cycle, the byproducts of which are deleterious reactive oxygen species. Beside this peculiarity, it is a target for numerous drugs and natural compounds such as melatonin, chloroquine, imiquimod, resveratrol, piceatannol, quercetin, and other flavonoids. Most of these enzyme-ligand interactions have been documented by numerous crystallographic studies, and now NQO2 is one of the best represented proteins in the structural biology database. Despite evidence for a causative role in several important diseases, the functional role of NQO2 remains poorly explored. In the present review, we aimed at detailing the main characteristics of NQO2 from a molecular pharmacology perspective. By drawing a clear border between facts and speculations, we hope to stimulate the future research toward a better understanding of this intriguing drug target. SIGNIFICANCE STATEMENT: Evidence is reviewed on the prevalent toxifying function of N-ribosyldihydronicotinamide:quinone oxidoreductase 2 while catalyzing the reduction of ortho-quinones such as dopamine quinone. The product of this reaction is unstable and generates a futile but harmful cycle (substrate/product/substrate) associated with reactive oxygen species generation.

Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments.Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as HisThese results demonstrated a relevance of Asp156 in the interaction of AcuI from E. coli K-12 with NADH as a coenzyme. These findings may provide reference information for shifting coenzyme specificity of acrylyl-CoA reductases.

The formation of memory for a novel experience is a critical cognitive capacity. The ability to form novel memories is sensitive to age-related pathologies and disease, to which prolonged metabolic stress is a major contributing factor. Presently, we describe a dopamine-dependent redox modulation pathway within the hippocampus of male mice that promotes memory consolidation. Namely, following novel information acquisition, quinone reductase 2 (QR2) is suppressed by miRNA-182 (miR-182) in the CA1 region of the hippocampus via dopamine D1 receptor (D1R) activation, a process largely facilitated by locus coeruleus activity. This pathway activation reduces ROS generated by QR2 enzymatic activity, a process that alters the intrinsic properties of CA1 interneurons 3 h following learning, in a form of oxidative eustress. Interestingly, novel experience decreases QR2 expression predominately in inhibitory interneurons. Additionally, we find that in aged animals this newly described QR2 pathway is chronically under activated, resulting in miR-182 underexpression and QR2 overexpression. This leads to accumulative oxidative stress, which can be seen in CA1 via increased levels of oxidized, inactivated potassium channel Kv2.1, which undergoes disulfide bridge oligomerization. This newly described interneuron-specific molecular pathway lies alongside the known mRNA translation-dependent processes necessary for long-term memory formation, entrained by dopamine in CA1. It is a process crucial for the distinguishing features of novel memory, and points to a promising new target for memory enhancement in aging and age-dependent diseases.SIGNIFICANCE STATEMENTOne way in which evolution dictates which sensory information will stabilize as an internal representation, relies on information novelty. Dopamine is a central neuromodulator involved in this process in the mammalian hippocampus. Here, we describe for the first time a dopamine D1 receptor-dependent quinone reductase 2 pathway in interneurons. This is a targeted redox event necessary to delineate a novel experience to a robust long-term internal representation. Activation of this pathway alone can explain the effect novelty has on “flashbulb” memories, and it can become dysfunctional with age and diseases, such as Alzheimer's disease.

NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architecture of the quinone-access channel in the enzyme has been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and the 39-kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.

Sulfur-oxidizing bacteria that are halophilic and acidophilic have gained interest because of their potential use in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH, which was previously identified as Acidithiobacillus thiooxidans based on its 16S rRNA gene sequence, is a chemolithoautotrophic marine bacterium exhibiting sodium chloride-stimulated thiosulfate-oxidizing activities. A novel thiosulfate:quinone oxidoreductase from strain SH (SH-TQO) has been purified from its solubilized membrane fraction. The gene for SH-TQO was determined from the draft genome sequence of the strain SH. Amino acid sequences of peptides generated by the in-gel trypsin digestion of SH-TQO were found in a protein encoded by locus tag B1757_09800 of the genome of the strain SH. The gene encoded 444 amino acids with a signal peptide of 29 amino acids and was annotated to encode a porin. The gene was located in a unique genomic region, not found in A. thiooxidans strains, suggesting that the strain SH acquired this region through a horizontal gene transfer. A protein–protein basic local alignment search revealed that sulfur-oxidizing bacteria, such as Acidithiobacillus species have proteins homologous to SH-TQO, though the degree of homologies was relatively low. The protein, DoxXA, which is homologous to TQO from Acidianus amvibalens, was also found in the genomic region.