Glycyl Radical Enzymes and Sulfonate Metabolism in the Microbiome.

Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C-S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C-S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome, where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H2S, a process linked to certain chronic diseases and conditions.

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Learning from the worm: the effectiveness of protein-bound Moco to treat Moco deficiency.

Molybdenum cofactor (Moco) is synthesized endogenously in humans and is essential for human development. Supplementation of Moco or its precursors has been explored as a therapy to treat Moco-deficient patients but with significant limitations. By using the nematode C. elegans as a model, Warnhoff and colleagues (pp. 212-217) describe the beneficial impact of protein-bound Moco supplementation to treat Moco deficiency. If such an effect is conserved, this advance from basic research in worms may have significant clinical implications as a novel therapy for molybdenum cofactor deficiency.

DsrMKJOP is the terminal reductase complex in anaerobic sulfate respiration.

Microbial dissimilatory sulfate reduction (DSR) is a key process in the Earth biogeochemical sulfur cycle. In spite of its importance to the sulfur and carbon cycles, industrial processes, and human health, it is still not clear how reduction of sulfate to sulfide is coupled to energy conservation. A central step in the pathway is the reduction of sulfite by the DsrAB dissimilatory sulfite reductase, which leads to the production of a DsrC-trisulfide. A membrane-bound complex, DsrMKJOP, is present in most organisms that have DsrAB and DsrC, and its involvement in energy conservation has been inferred from sequence analysis, but its precise function was so far not determined. Here, we present studies revealing that the DsrMKJOP complex of the sulfate reducer Archaeoglobus fulgidus works as a menadiol:DsrC-trisulfide oxidoreductase. Our results reveal a close interaction between the DsrC-trisulfide and the DsrMKJOP complex and show that electrons from the quinone pool reduce consecutively the DsrM hemes b, the DsrK noncubane [4Fe-4S]3+/2+ catalytic center, and finally the DsrC-trisulfide with concomitant release of sulfide. These results clarify the role of this widespread respiratory membrane complex and support the suggestion that DsrMKJOP contributes to energy conservation upon reduction of the DsrC-trisulfide in the last step of DSR.

A ratiometric fluorescence probe for bisulfite detection in live cells and meat samples.

The development of reliable probe technology for the detection of bisulfite (HSO3-) in situ in food and biological samples is contributing significantly to food quality and safety assurance as well as community health. In this work, a responsive probe, EHDI, is developed for ratiometric fluorescence detection of HSO3- in aqueous solution, meat samples, and living cells. The probe is designed based on the HSO3- triggered 1,4-addition of electron deficit C = C bond of EHDI. As a result of this specific 1,4-addition, the π-conjugation system was destructed, resulting in blue shifts of the emission from 687 to 440 nm and absorption from 577 to 355 nm. The probe has good water solubility, high sensitivity and selectivity, allowing it to be used for imaging of HSO3- internalization and production endogenously. The capability of probe EHDI for HSO3- was then validated by traditional HPLC technology, enabling accurately detect HSO3- in beef samples. The successful development of this probe thus offers a new tool for investigating HSO3- in situ in food and biological conditions.

Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

The DsrD functional marker protein is an allosteric activator of the DsrAB dissimilatory sulfite reductase.

Dissimilatory sulfur metabolism was recently shown to be much more widespread among bacteria and archaea than previously believed. One of the key pathways involved is the dsr pathway that is responsible for sulfite reduction in sulfate-, sulfur-, thiosulfate-, and sulfite-reducing organisms, sulfur disproportionators and organosulfonate degraders, or for the production of sulfite in many photo- and chemotrophic sulfur-oxidizing prokaryotes. The key enzyme is DsrAB, the dissimilatory sulfite reductase, but a range of other Dsr proteins is involved, with different gene sets being present in organisms with a reductive or oxidative metabolism. The dsrD gene codes for a small protein of unknown function and has been widely used as a functional marker for reductive or disproportionating sulfur metabolism, although in some cases this has been disputed. Here, we present in vivo and in vitro studies showing that DsrD is a physiological partner of DsrAB and acts as an activator of its sulfite reduction activity. DsrD is expressed in respiratory but not in fermentative conditions and a ΔdsrD deletion strain could be obtained, indicating that its function is not essential. This strain grew less efficiently during sulfate and sulfite reduction. Organisms with the earliest forms of dsrAB lack the dsrD gene, revealing that its activating role arose later in evolution relative to dsrAB.

Isethionate is an intermediate in the degradation of sulfoacetate by the human gut pathobiont Bilophila wadsworthia.

The obligately anaerobic sulfite-reducing bacterium Bilophila wadsworthia is a common human pathobiont inhabiting the distal intestinal tract. It has a unique ability to utilize a diverse range of food- and host-derived sulfonates to generate sulfite as a terminal electron acceptor (TEA) for anaerobic respiration, converting the sulfonate sulfur to H2S, implicated in inflammatory conditions and colon cancer. The biochemical pathways involved in the metabolism of the C2 sulfonates isethionate and taurine by B. wadsworthia were recently reported. However, its mechanism for metabolizing sulfoacetate, another prevalent C2 sulfonate, remained unknown. Here, we report bioinformatics investigations and in vitro biochemical assays that uncover the molecular basis for the utilization of sulfoacetate as a source of TEA (STEA) for B. wadsworthia, involving conversion to sulfoacetyl-CoA by an ADP-forming sulfoacetate-CoA ligase (SauCD), and stepwise reduction to isethionate by NAD(P)H-dependent enzymes sulfoacetaldehyde dehydrogenase (SauS) and sulfoacetaldehyde reductase (TauF). Isethionate is then cleaved by the O2-sensitive isethionate sulfolyase (IseG), releasing sulfite for dissimilatory reduction to H2S. Sulfoacetate in different environments originates from anthropogenic sources such as detergents, and natural sources such as bacterial metabolism of the highly abundant organosulfonates sulfoquinovose and taurine. Identification of enzymes for anaerobic degradation of this relatively inert and electron-deficient C2 sulfonate provides further insights into sulfur recycling in the anaerobic biosphere, including the human gut microbiome.

Pharmacodynamic profiling in three patients with molybdenum cofactor deficiency type A reveals prolonged biological effects after withdrawal of cyclic pyranopterin monophosphate.

Molybdenum cofactor deficiency type A has successfully been treated in a small number of children with daily intravenous administration of cyclic pyranopterin monophosphate. Pharmacodynamic data for this novel treatment have not been published and alternative dosing intervals have not been explored. We monitored pharmacodynamic biomarkers of sulfite oxidase and xanthine oxidoreductase activity in three patients with MoCD-A for a period of 2 to 9 months after discontinuation of cPMP substitution. We found that the clinical and metabolic effects were sustained for longer than expected, over 7 days at least. Our data implicate a biological half-life of the molybdenum cofactor dependent enzyme activities of approximately 3 days and suggest the possibility that less frequent than once daily dosing intervals could be a safe alternative to current practice.

Phylogeny and evolution of dissimilatory sulfite reduction in prokaryotes.

Sulfate is the second most common nonmetallic ion in modern oceans, as its concentration dramatically increased alongside tectonic activity and atmospheric oxidation in the Proterozoic. Microbial sulfate/sulfite metabolism, involving organic carbon or hydrogen oxidation, is linked to sulfur and carbon biogeochemical cycles. However, the coevolution of microbial sulfate/sulfite metabolism and Earth's history remains unclear. Here, we conducted a comprehensive phylogenetic analysis to explore the evolutionary history of the dissimilatory sulfite reduction (Dsr) pathway. The phylogenies of the Dsr-related genes presented similar branching patterns but also some incongruencies, indicating the complex origin and evolution of Dsr. Among these genes, dsrAB is the hallmark of sulfur-metabolizing prokaryotes. Our detailed analyses suggested that the evolution of dsrAB was shaped by vertical inheritance and multiple horizontal gene transfer events and that selection pressure varied across distinct lineages. Dated phylogenetic trees indicated that key evolutionary events of dissimilatory sulfur-metabolizing prokaryotes were related to the Great Oxygenation Event (2.4-2.0 Ga) and several geological events in the "Boring Billion" (1.8-0.8 Ga), including the fragmentation of the Columbia supercontinent (approximately 1.6 Ga), the rapid increase in marine sulfate (1.3-1.2 Ga), and the Neoproterozoic glaciation event (approximately 1.0 Ga). We also proposed that the voluminous iron formations (approximately 1.88 Ga) might have induced the metabolic innovation of iron reduction. In summary, our study provides new insights into Dsr evolution and a systematic view of the coevolution of dissimilatory sulfur-metabolizing prokaryotes and the Earth's environment.

Reaction Mechanism Catalyzed by the Dissimilatory Sulfite Reductase - The Role of the Siroheme-[4FeS4] Cofactor.

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant.

Consensus guidelines for the diagnosis and management of isolated sulfite oxidase deficiency and molybdenum cofactor deficiencies.

Sulfite intoxication is the hallmark of four ultrarare disorders that are caused by impaired sulfite oxidase activity due to genetic defects in the synthesis of the molybdenum cofactor or of the apoenzyme sulfite oxidase. Delays on the diagnosis of these disorders are common and have been caused by their unspecific presentation of acute neonatal encephalopathy with high early mortality, followed by the evolution of dystonic cerebral palsy and also by the lack of easily available and reliable diagnostic tests. There is significant variation in survival and in the quality of symptomatic management of affected children. One of the four disorders, molybdenum cofactor deficiency type A (MoCD-A) has recently become amenable to causal treatment with synthetic cPMP (fosdenopterin). The evidence base for the rational use of cPMP is very limited. This prompted the formulation of these clinical guidelines to facilitate diagnosis and support the management of patients. The guidelines were developed by experts in diagnosis and treatment of sulfite intoxication disorders. It reflects expert consensus opinion and evidence from a systematic literature search.

Rapid Oxidative Dissolution of Zerovalent Iron Induced by Sulfite for Efficient Removal of Arsenate and Arsenite: Selective Formation of Scorodite.

In this study, we proposed a moderate oxidation strategy for accelerating the oxidative dissolution of zerovalent iron (ZVI) using sulfite (S(IV)), thereby improving the removal of As(V) and As(III). Results revealed that, in the presence of 2.0 mM S(IV), both As(V) and As(III) were selectively converted into scorodite at pH0 3.0-7.0, while As(III) oxidation and As(V) immobilization were impressed over pH0 8.0-10.0. Batch experiments, radical quenching experiments, and electron spin resonance (ESR) measurements demonstrated that ZVI initially boosted S(IV) activation to generate SO4•-, •OH, and protons, and in turn, ZVI was further oxidized more intensely by these radicals than by oxygen. Concurrently, substantial protons derived from S(IV) oxidation neutralized hydroxyls produced by ZVI oxidation, maintaining an acidic environment conducive to the generation of scorodite rather than iron (hydr)oxides. Characterizations of X-ray diffraction (XRD), Raman, attenuated total reflectance-Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), X-ray absorption fine structure (XAFS), field emission scanning electron microscopy (FESEM), and high-resolution transmission electron microscopy (HRTEM) confirmed that scorodite was formed in situ and then exfoliated from the surface of ZVI, and approximately 75% of ZVI could still be recovered, which contributed to efficient As removal in successive runs and real As-polluted wastewater. The application of S(IV) achieved a balance among ZVI reactivity improvement, As(V)/As(III) removal, and raw material consumption, making it a promising approach for addressing arsenic contamination in wastewater treatment.

Insight into the Efficient Selective Reduction of Cr(VI) in Sulfite/UV Process under Near-Neutral Conditions: The Critical Role of In Situ-Generated Sulfite Radical.

Efficient removal of contaminants in complex water matrices under mild conditions is highly desirable but still challenging. In this study, we unraveled the overlooked but crucial role of sulfite radical (SO3·-) in the efficient selective reduction of toxic Cr(VI) under near-neutral conditions. Fast removal of Cr(VI) at around pH 7 in sulfite/UV was found to be attributable to high reactivity of SO3·- toward HCrO4- (∼5.3 × 106 M-1 s-1). Furthermore, SO3·- was fast generated in situ via one-electron oxidation of S(IV) by transient reactive protonated Cr(V) and Cr(IV) intermediates. Therefore, the specific reactivity of SO3·- and its in situ generation together resulted in the surprisingly positive effect of nitrate and the efficient reduction of Cr(VI) in authentic surface water and industrial wastewater. A mathematical model was developed to simulate Cr(VI) removal in the process, and thus quantitatively demonstrated the roles of reactive species, i.e., SO3·- contributed to ∼93% of Cr(VI) reduction in surface water. Overall, this study provides an insight into the pivotal role of SO3·- in Cr(VI) reduction, and underscores its significance in selective reduction and detoxification of contaminants.

Kinetic Model for Predicting Perfluoroalkyl Acid Degradation During UV-Sulfite Treatment.

Hydrated electron (eaq-) treatment processes show great potential in remediating recalcitrant water contaminants, including perfluoroalkyl and polyfluoroalkyl substances (PFAS). However, treatment efficacy depends upon many factors relating to source water composition, UV light source characteristics, and contaminant reactivity. Here, we provide critical insights into the complex roles of solution parameters on contaminant abatement through application of a UV-sulfite kinetic model that incorporates first-principles information on eaq- photogeneration and reactivity. The model accurately predicts decay profiles of short-chain perfluoroalkyl acids (PFAAs) during UV-sulfite treatment and facilitates quantitative interpretation of the effects of changing solution composition on PFAS degradation rates. Model results also confirm that the enhanced degradation of PFAAs observed under highly alkaline pH conditions results from changes in speciation of nontarget eaq- scavengers. Reverse application of the model to UV-sulfite data collected for longer chain PFAAs enabled estimation of bimolecular rate constants (k2, M-1 s-1), providing an alternative to laser flash photolysis (LFP) measurements that are not feasible due to the water solubility limitations of these compounds. The proposed model links the disparate means of investigating eaq- processes, namely, UV photolysis and LFP, and provides a framework to estimate UV-sulfite treatment efficacy of PFAS in diverse water sources.

A FRET-based multifunctional fluorescence probe for the simultaneous detection of sulfite and viscosity in living cells.

Viscosity and sulfur dioxide derivatives were significant indicators for the assessment of health threat and even cancers, therefore, on-site and real time detection of viscosity and sulfur dioxide derivatives has obtained considerable attentions. An FRET-based fluorescence probe JZX was designed and synthesized based on a novel energy donor of N,N-diethyl-4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzamide fluorophore. JZX exhibited a large Stokes shift (230 nm), high energy transfer efficiency, wide emission channel gap (135 nm) and excellent stability and biocompatibility. JZX detected sulfur dioxide with low detection limit (55 nM), fast responding (16 min), high selectivity and sensitivity. Additionally, JZX tend to target endoplasmic reticulum of which normal metabolism will be disturbed by the abnormal levels of viscosity and sulfur dioxide derivatives. Prominently, JZX could concurrently detect viscosity and sulfur dioxide derivatives depending on different fluorescence signals in living cells for the screening of cancer cells. Hence, probe JZX will be a promising candidate for the detection of viscosity and sulfur dioxide derivatives, and even for the diagnosis of liver cancers.

Design and application of a novel "turn-on" fluorescent probe for imaging sulfite in living cells and inflammation models.

Sulfite is one of the main existing forms of sulfur dioxide (SO2) in living system, which has been recognized as an endogenous mediator in inflammation. Evidence has accumulated to show that abnormal level of sulfite is associated with many inflammatory diseases, including neurological diseases and cancers. Herein, a novel fluorescent probe named QX-OA was designed and synthesized to detect sulfite. QX-OA was constructed by choosing quinolinium-xanthene as the fluorophore and levulinate as the specific and relatively steady recognition reaction. The probe showed remarkable green turn-on signal at 550 nm, together with high sensitivity (90-fold) and excellent selectivity to sulfite over other possible interfering species. In the meantime, QX-OA was successfully applied to visualize endogenous and exogenous sulfite in Hela cells. In the LPS-induced inflammation model, QX-OA could visualize the dose-dependent increase of sulfite level (0-2 mg/mL). Consequently, QX-OA was determined to be a potential method for detecting sulfite in pre-clinical diagnosis.

Role of reactive manganese and oxygen species in the KMnO4/Na2SO3 process for purification of algal-rich water and membrane fouling alleviation.

Ultrafiltration (UF) is a highly efficient technique for algal-rich water purification, but it is heavily contaminated due to the complex water characteristics. To solve this problem, potassium permanganate (KMnO4) oxidation enhanced with sodium sulfite (Na2SO3) was proposed as a pretreatment means. The results showed that the end-normalized flux was elevated from 0.10 to 0.91, and the reversible fouling resistance was reduced by 99.95%. The membrane fouling mechanism also changed obviously, without the generation of cake filtration. Regarding the properties of algal-rich water, the zeta potential was decreased from -29.50 to -5.87 mV after KMnO4/Na2SO3 pretreatment, suggesting that the electrostatic repulsion was significantly reduced. Meanwhile, the fluorescent components in algal-rich water were significantly eliminated, and the removal of dissolved organic carbon was increased to 67.46%. In the KMnO4/Na2SO3 process, reactive manganese species (i.e., Mn(V), Mn(III) and MnO2) and reactive oxygen species (i.e., SO4•- and •OH) played major roles in purifying algal-rich water. Specifically, SO4•-, •OH, Mn(V) and Mn(III) could effectively oxidize algal pollutants. Simultaneously, the in-situ adsorption and coagulation of MnO2 could accelerate the formation of flocs by decreasing the electrostatic repulsion between cells, and protect the algal cells from being excessive oxidized. Overall, the KMnO4/Na2SO3 process showed significant potential for membrane fouling alleviation in purifying algal-rich water.

Promoting effect of TiVC MXene on cathodic electrogenerated chemiluminescence of Ru(bpy)32+ and its application in the sensitive detection of sulfite.

The enhanced cathodic ECL of Ru(bpy)32+ at a bimetallic element MXenes (TiVC MXene) modified electrode in neutral aqueous condition is reported. TiVC MXene significantly catalyzed the oxygen reduction reaction (ORR) as well as the electrochemical reduction of Ru(bpy)32+ to produce reactive oxygen species and Ru(bpy)3+. The obtained hydroxyl radical (OH∙) not only oxidized Ru(bpy)3+ to generate Ru(bpy)32+* and emit light through coreactant pathway, but also oxidized Ru(bpy)32+ to Ru(bpy)33+, which caused an annihilation ECL reaction. As a result, two pathways occurred simultaneously to generate strong cathodic ECL signal. Sulfite removes the dissolved oxygen in water and reduces the occurrence of ORR, which prohibits the generation of OH∙ to decrease the ECL signal. The decrement of ECL intensity varied linearly with the concentration of sulfite in the range 2 nM to 50 µM with a detection limit of 0.14 nM (3σ). The proposed sensor exhibited good analytical performance, and could be used in the detection of sulfite in real samples. The results revealed that the electrocatalytic behavior of TiVC MXene is the key factor for strong cathodic Ru(bpy)32+ ECL, which provides new application in ECL sensing field.

Integrating UPLC-QTOF-MS/MS and UPLC-DAD to evaluate the influence of sulfur-fumigated Paeoniae Radix Alba on the overall quality of three Si-Wu-Tang formulations.

INTRODUCTION: Sulfur-fumigation of Paeoniae Radix Alba (PRA) could induce the chemical transformation of its bioactive component paeoniflorin into a sulfur-containing derivative paeoniflorin sulfite, and thus alter the quality, bioactivities, pharmacokinetics, and toxicities of PRA. However, how sulfur-fumigated PRA (S-PRA) affects the quality of PRA-containing complex preparations has not been intensively evaluated. OBJECTIVES: We intend to evaluate the influence of S-PRA on the overall quality of three kinds of Si-Wu-Tang (SWT) formulations, i.e., decoction (SWT-D), granule (SWT-G), and mixture (SWT-M). MATERIAL AND METHODS: An UPLC-DAD multi-components quantification method was used to compare the transfer rates of paeoniflorin sulfite and other 10 bioactive components between S-PRA-containing and NS-PRA-containing SWT formulations. An UPLC-QTOF-MS/MS-based target metabolomics approach was applied to explore the differential sulfur-containing derivatives in S-PRA-containing SWT formulations. RESULTS: The transfer rates of paeoniflorin sulfite in three S-PRA-containing SWT formulations were all higher than 100%. Moreover, S-PRA also increased the transfer rate of 5-hydroxymethylfurfural, 1,2,3,4,6-O-pentagalloylglucose, whereas decreased that of paeoniflorin, albiflorin, and ferulic acid in three SWT formulations. Six pinane monoterpene glucoside sulfites originally identified in S-PRA, were also detectable in three S-PRA-containing SWT formulations. In addition, seven phenolic acid sulfites including (3Z)-6-sulfite-ligustilide, (3E)-6-sulfite-ligustilide, 6,8-disulfite-ligustilide, ferulic acid sulfite, neochlorogenic acid sulfite, chlorogenic acid sulfite, and angelicide sulfite (or isomer) were newly identified in these three S-PRA-containing formulations. CONCLUSION: S-PRA could differentially affect the transfer rate of paeoniflorin sulfite and other bioactive components during the preparation of three SWT formulations and subsequently the overall quality thereof.