Paramagnetic NMR to study iron sulfur proteins: 13C detected experiments illuminate the vicinity of the metal center.

The robustness of NMR coherence transfer in proximity of a paramagnetic center depends on the relaxation properties of the nuclei involved. In the case of Iron-Sulfur Proteins, different pulse schemes or different parameter sets often provide complementary results. Tailored versions of HCACO and CACO experiments significantly increase the number of observed Cα/C' connectivities in highly paramagnetic systems, by recovering many resonances that were lost due to paramagnetic relaxation. Optimized 13C direct detected experiments can significantly extend the available assignments, improving the overall knowledge of these systems. The different relaxation properties of Cα and C' nuclei are exploited in CACO vs COCA experiments and the complementarity of the two experiments is used to obtain structural information. The two [Fe2S2]+ clusters containing NEET protein CISD3 and the one [Fe4S4]2+ cluster containing HiPIP protein PioC have been taken as model systems. We show that tailored experiments contribute to decrease the blind sphere around the cluster, to extend resonance assignment of cluster bound cysteine residues and to retrieve details on the topology of the iron-bound ligand residues.

The impact of genotype on the phenotype of Mycobacterium tuberculosis ΔsufR mutants.

Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the operon encoding the primary FeS cluster biogenesis system. Previously, three independently isolated mutants (ΔRv1460stop_1.19, ΔRv1460stop _5.19 and ΔRv1460stop _5.20) harbouring the same deletion in sufR, displayed different growth kinetics in OADC supplemented 7H9 media. To investigate this discrepancy, we performed whole genome sequencing of the 3 mutants and the wild-type progenitor. Single nucleotide polymorphisms (SNPs) were identified in 3 genes in the ΔRv1460stop_1.19 mutant and one gene in the ΔRv1460stop_5.20 mutant. Phenotyping of the ΔRv1460stop_5.19 mutant, which had no additional SNPs, revealed increased susceptibility to clofazimine, DMNQ and menadione, while uptake and survival in THP-1 cells were not significantly different from the wild-type strain. Given that these results differ from those reported for other sufR deletion mutants (ΔSufRMTB and MtbΔSufR), they suggest that the position of the sufR deletion and the genotype of the progenitor strain impact the resulting phenotype.

The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli.

Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.

Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli.

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E. coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E. coli Fur also binds a [2Fe-2S] cluster in wildtype E. coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box in vitro, and are inactive to complement the function of Fur in vivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E. coli cells.

Ferrodifferentiation regulates neurodevelopment via ROS generation.

Iron is important for life, and iron deficiency impairs development, but whether the iron level regulates neural differentiation remains elusive. In this study, with iron-regulatory proteins (IRPs) knockout embryonic stem cells (ESCs) that showed severe iron deficiency, we found that the Pax6- and Sox2-positive neuronal precursor cells and Tuj1 fibers in IRP1-/-IRP2-/- ESCs were significantly decreased after inducing neural differentiation. Consistently, in vivo study showed that the knockdown of IRP1 in IRP2-/- fetal mice remarkably affected the differentiation of neuronal precursors and the migration of neurons. These findings suggest that low intracellular iron status significantly inhibits neurodifferentiation. When supplementing IRP1-/-IRP2-/- ESCs with iron, these ESCs could differentiate normally. Further investigations revealed that the underlying mechanism was associated with an increase in reactive oxygen species (ROS) production caused by the substantially low level of iron and the down-regulation of iron-sulfur cluster protein ISCU, which, in turn, affected the proliferation and differentiation of stem cells. Thus, the appropriate amount of iron is crucial for maintaining normal neural differentiation that is termed ferrodifferentiation.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

X-ray absorption spectroscopy of copper and iron in sheep digesta.

BACKGROUND: The bioavailable supply of copper to ruminants has long been problematic. Complexities in supply exist due to interactions with other dietary elements in the rumen, most notably with iron or molybdenum in combination with sulphur, which can result in copper binding preventing its absorption. The molybdenum-sulphur-copper interaction has been extensively studied over the years. However, very little is known about the iron-sulphur-copper interaction, especially its mode of action in the gastrointestinal tract. METHODS: In the present work digesta from the rumen and jejunum of sheep fed a high copper, sulphur and iron diet was analysed using X-ray absorption spectroscopy (XAS). RESULTS: X-ray absorption fine structure (XAFS) and X-ray absorption near edge structure (XANES) indicated that all of the copper and iron had changed in bonding in the rumen and that the oxidation state of the elements had been reduced into a mix of Fe2+ & Fe3+ and Cu+ with some Cu0. CONCLUSION: The copper compounds were most likely to be thiol co-ordinated in line with Cu+ chemistry. Changes to the copper compounds took place in the jejunum, although thiols were still highly favoured the possible existence of a copper-iron-sulphur complex which also included oxygen and chloride was also observed. This possibly has some resemblance to the crystal structure of bornite.

Hypoketotic hypoglycemia without neuromuscular complications in patients with SLC25A32 deficiency.

Mitochondrial flavin adenine dinucleotide (FAD) transporter deficiencies are new entities recently reported to cause a neuro-myopathic phenotype. We report three patients from two unrelated families who presented primarily with hypoketotic hypoglycemia. They all had acylcarnitine profiles suggestive of multiple acyl-CoA dehydrogenase deficiency (MADD) with negative next-generation sequencing of electron-transfer flavoprotein genes (ETFA, ETFB, and ETFDH). Whole exome sequencing revealed a homozygous c.272 G > T (p.Gly91Val) variant in exon 2 of the SLC25A32 gene. The three patients shared the same variant, and they all demonstrated similar clinical and biochemical improvement with riboflavin supplementation. To date, these are the first patients to be reported with hypoketotic hypoglycemia without the neuromuscular phenotype previously reported in patients with SLC25A32 deficiency.

Clinical, pathological and genetic features and follow-up of 110 patients with late-onset MADD: a single-center retrospective study.

To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy.

Intrinsically Disordered Region Modulates Ligand Binding in Glutaredoxin 1 from Trypanosoma Brucei.

Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron-sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.

A conserved and seemingly redundant Escherichia coli biotin biosynthesis gene expressed only during anaerobic growth.

Biotin is an essential metabolic cofactor and de novo biotin biosynthetic pathways are widespread in microorganisms and plants. Biotin synthetic genes are generally found clustered into bio operons to facilitate tight regulation since biotin synthesis is a metabolically expensive process. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio operon gene bioD. Several studies have reported transcriptional activation of ynfK a gene of unknown function, under anaerobic conditions. Alignments of YnfK with BioD have led to suggestions that YnfK has DTBS activity. We report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. We confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria.

ETF dehydrogenase advances in molecular genetics and impact on treatment.

Electron transfer flavoprotein dehydrogenase, also called ETF-ubiquinone oxidoreductase (ETF-QO), is a protein localized in the inner membrane of mitochondria, playing a central role in the electron-transfer system. Indeed, ETF-QO mediates electron transport from flavoprotein dehydrogenases to the ubiquinone pool. ETF-QO mutations are often associated with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD, OMIM#231680), a multisystem genetic disease characterized by various clinical manifestations with different degrees of severity. In this review, we outline the clinical features correlated with ETF-QO deficiency and the benefits obtained from different treatments, such as riboflavin, L-carnitine and/or coenzyme Q10 supplementation, and a diet poor in fat and protein. Moreover, we provide a detailed summary of molecular and bioinformatic investigations, describing the mutations identified in ETFDH gene and highlighting their predicted impact on enzymatic structure and activity. In addition, we report biochemical and functional analysis, performed in HEK293 cells and patient fibroblasts and muscle cells, to show the relationship between the nature of ETFDH mutations, the variable impairment of enzyme function, and the different degrees of RR-MADD severity. Finally, we describe in detail 5 RR-MADD patients carrying different ETFDH mutations and presenting variable degrees of clinical symptom severity.

Proteomic Analysis of Proteins Associated with Inhibition of Pseudomonas aeruginosa Resistance to Imipenem Mediated by the Chinese Herbal Medicine Qi Gui Yin.

Objective: Antibiotic resistance of Pseudomonas aeruginosa (PA) that lowers the effectiveness of current treatments for pneumonia is a growing problem. Qi Gui Yin is a Chinese herbal medicine that has been used to improve the efficacy of antibiotic therapy against antibiotic-resistant bacteria. This study aimed to elucidate the mechanism by which Qi Gui Yin inhibits antibiotic resistance of PA. Methods: Active components of Qi Gui Yin were analyzed by chromatography. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) technology was used to compare protein expression profiles of PA strains cultured in serum from rats that were and were not treated with Qi Gui Yin. Quantitative polymerase chain reaction (qPCR) analysis was performed to detect gene expression changes. Results: Proteomic analysis identified 76 differentially expressed proteins between PA strains cultured in serum from rats that were or were not treated with Qi Gui Yin. Bioinformatics analysis revealed that the largest number of differentially expressed proteins were associated with resistance mechanisms such as quorum sensing, bacterial biofilm formation, and active pumping. In addition, qPCR analysis confirmed that downregulation of iscU and arcA gene expression was associated with Qi Gui Yin treatment. Conclusions: Serum from Qi Gui Yin-treated rats could effectively inhibit antibiotic resistance of PA. Chlorogenic acid and astragaloside IV are the main components of Qi Gui Yin, which may mediate inhibition of antibiotic resistance. Our findings provide new insights into strategies involving Chinese herbal medicine that can be used to treat pneumonia caused by antibiotic-resistant bacteria.

Unconventional Biocatalytic Approaches to the Synthesis of Chiral Sulfoxides.

Sulfoxides are a class of organic compounds that find wide application in medicinal and organic chemistry. Several biocatalytic approaches have been developed to synthesise enantioenriched sulfoxides, mainly by exploiting oxidative enzymes. Recently, the use of reductive enzymes such as Msr and Dms has emerged as a new, alternative method to obtain enantiopure sulfoxides from racemic mixtures. In parallel, novel oxidative approaches, employing nonclassical solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), have been developed as greener and more sustainable biocatalytic synthetic pathways. This minireview aims highlights the recent advances made in the biocatalytic synthesis of enantioenriched sulfoxides by employing such unconventional approaches.

Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

S. aureus USA300 isolates utilize the copBL and copAZ gene products to prevent Cu intoxication. We created and examined a ΔcopAZ ΔcopBL mutant strain (cop-). The cop- strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in the cop- background and isolated strains with Tn insertions in the mntABC operon that permitted growth in the presence of Cu. The mutations were in mntA and they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing a mntA::Tn accumulated less Cu than the parent strain. Mn(II) supplementation improved growth when cop- was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor of mntABC transcription. A ΔmntR strain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction of mntA::Tn. Over-expression of mntABC increased cellular Cu load and sensitivity to Cu. The presence of a mntA::Tn mutation protected iron-sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.

DFT Study on the Mechanism of Iron-Catalyzed Diazocarbonylation.

The mechanism of the carbonylation of diazomethane in the presence of iron-carbonyl-phosphine catalysts has been investigated by means of DFT calculations at the M06/def-TZVP//B97D3/def2-TZVP level of theory, in combination with the SMD solvation method. The reaction rate is determined by the formation of the coordinatively unsaturated doublet-state Fe(CO)3(P) precursor followed by the diazoalkane coordination and the N2 extrusion. The free energy of activation is predicted to be 18.5 and 28.2 kcal/mol for the PF3 and PPh3 containing systems, respectively. Thus, in the presence of less basic P-donor ligands with stronger π-acceptor properties, a significant increase in the reaction rate can be expected. According to energy decomposition analysis combined with natural orbitals of chemical valence (EDA-NOCV) calculations, diazomethane in the Fe(CO)3(phosphine)(η1-CH2N2) adduct reveals a π-donor-π-acceptor type of coordination.

Clinical characteristics and gene mutation analysis of an adult patient with ETFDH­related multiple acyl­CoA dehydrogenase deficiency.

Multiple acyl­CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive disorder of fatty acid metabolism caused by defects in electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETFDH). These defects are mainly classified into the neonatal and late­onset types, based on their clinical manifestations. ETFDH gene mutations are generally considered to be associated with the late­onset type. The present study reported an adult woman with late­onset MADD accompanied with biochemical and muscle biopsy findings indicating metabolic disorders. Gene sequencing analysis showed that the c.1514T>C homozygous mutation in the region of the 12th exon of the ETFDH gene, which led to the amino acid substitution p.I505T (isoleucine > threonine), resulting in defective ETFDH protein function. The results of family verification revealed that the homozygous mutation originated from her parents. The female patient was treated with a large dose of vitamin B2, L­carnitine and coenzyme Q10, and the symptoms were significantly relieved. The c.1514T>C mutation in the ETFDH gene, was considered as a novel pathogenic mutation that had not been previously reported. Therefore, it was hypothesized that this mutation was responsible for the clinical characteristics of the adult female patient. Overall, this novel mutation could expand the spectrum of the ETFDH gene mutation and provide the basis for the etiological and prenatal diagnosis of MADD.

Neonatal-onset multiple acyl-CoA dehydrogenase deficiency (MADD) in the ETFDH gene: A case report and a literature review.

RATIONALE: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting fatty acid, amino acid, and choline metabolism. The clinical manifestation of MADD is heterogeneous, from severe neonatal forms to mild late-onset forms. PATIENT CONCERNS: Here, we report a patient who presented with severe hypoglycemia and exercise intolerance suggestive of MADD. Serum tandem mass spectrometry analysis indicated elevated levels of various acyl carnitines at 25 days of age. Exome sequencing of the proband revealed compound heterozygous mutations, c. 413T>G (p.Leu138Arg) and c.1667C > G (p.Pro556Arg), in the ETFDH gene as the probable causative mutations. DIAGNOSES: Based on the patient's clinical presentation and test results, the patient was diagnosed with MADD. INTERVENTIONS: A high-calorie and reduced-fat diet was given together with oral supplements of L-carnitine (150 mg/day). OUTCOMES: He passed away at the age of 4 months because of severe respiratory distress accompanied by muscle weakness. LESSONS: He passed away at the age of 4 months because of severe respiratory distress accompanied by muscle weakness. Clinicians should consider MADD in the differential diagnosis when patients present with muscle weakness and biochemical abnormalities. Gene testing plays a critical role in confirming the diagnosis of MADD and may not only prevent the need for invasive testing but also allow for timely initiation of treatment.

Methane, arsenic, selenium and the origins of the DMSO reductase family.

Mononuclear molybdoenzymes of the dimethyl sulfoxide reductase (DMSOR) family catalyze a number of reactions essential to the carbon, nitrogen, sulfur, arsenic, and selenium biogeochemical cycles. These enzymes are also ancient, with many lineages likely predating the divergence of the last universal common ancestor into the Bacteria and Archaea domains. We have constructed rooted phylogenies for over 1,550 representatives of the DMSOR family using maximum likelihood methods to investigate the evolution of the arsenic biogeochemical cycle. The phylogenetic analysis provides compelling evidence that formylmethanofuran dehydrogenase B subunits, which catalyze the reduction of CO2 to formate during hydrogenotrophic methanogenesis, constitutes the most ancient lineage. Our analysis also provides robust support for selenocysteine as the ancestral ligand for the Mo/W atom. Finally, we demonstrate that anaerobic arsenite oxidase and respiratory arsenate reductase catalytic subunits represent a more ancient lineage of DMSORs compared to aerobic arsenite oxidase catalytic subunits, which evolved from the assimilatory nitrate reductase lineage. This provides substantial support for an active arsenic biogeochemical cycle on the anoxic Archean Earth. Our work emphasizes that the use of chalcophilic elements as substrates as well as the Mo/W ligand in DMSORs has indelibly shaped the diversification of these enzymes through deep time.