RESUMEN
Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E0. Two of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.
Asunto(s)
Azotobacter vinelandii , Molibdoferredoxina , Nitrogenasa , Selenio , Azufre , Espectroscopía de Resonancia por Spin del Electrón/métodos , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/metabolismo , Nitrogenasa/metabolismo , Nitrogenasa/química , Molibdoferredoxina/metabolismo , Molibdoferredoxina/química , Selenio/metabolismo , Selenio/química , Azufre/metabolismo , Azufre/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/químicaRESUMEN
The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the variant surface glycoprotein or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory; minor alterations within these exposed epitopes confer antigenically distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross-reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.
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Trypanosoma brucei brucei , Trypanosoma , Animales , Epítopos Inmunodominantes , Evasión Inmune , Glicoproteínas Variantes de Superficie de Trypanosoma , Variación Antigénica , Epítopos , MamíferosRESUMEN
Two new populations of Pyrgulopsis were found in springs in the Chihuahuan desert region of Texas. Pyrgulopsis Call & Pilsbry, 1886 are a diverse group of snails occupying freshwater springs, usually narrow-range endemics limited to a single spring. Their small ranges confer substantial conservation concern as the drying or disturbance of a single spring can lead to their extinction. We use mitochondrial COI and nuclear LSU sequences, shell morphometrics, and anatomical features to distinguish a new species of Pyrgulopsis endemic to the Big Bend region of Texas.
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Gastrópodos , Animales , Agua DulceRESUMEN
A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.
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Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/ultraestructura , Proteínas de Drosophila/ultraestructura , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/ultraestructura , Proteínas de Unión al ARN/ultraestructura , Dispersión del Ángulo Pequeño , Factores de Transcripción/ultraestructura , Difracción de Rayos XRESUMEN
Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.
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Proteínas de Drosophila , Cinesinas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Transporte de ARN , ARN Mensajero/metabolismo , Tropomiosina/metabolismoRESUMEN
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.
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Benchmarking , Laboratorios , Calorimetría , Reproducibilidad de los Resultados , TemperaturaRESUMEN
Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
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Algoritmos , Estabilidad Proteica , Animales , Escherichia coli/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Solubilidad , Temperatura , Pez CebraRESUMEN
Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.
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Resistencia a Medicamentos/inmunología , Suramina/metabolismo , Trypanosoma brucei rhodesiense/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Variación Antigénica/efectos de los fármacos , Variación Antigénica/inmunología , Sitios de Unión , Cristalografía por Rayos X , Resistencia a Medicamentos/genética , Endocitosis/genética , Evasión Inmune , Mutación , Unión Proteica , Conformación Proteica , Suramina/toxicidad , Tripanocidas/metabolismo , Tripanocidas/toxicidad , Trypanosoma brucei rhodesiense/química , Trypanosoma brucei rhodesiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/metabolismo , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Pyrgulopsis Call Pilsbry, 1886 is a genus of small (<5 mm) spring snails, usually endemic to single freshwater springs. Two new populations of Pyrgulopsis found in very small, isolated springs and spring runs in the mainstem Rio Grande watershed of western Texas are distinguished from congeners. Mitochondrial and nuclear sequences, morphometrics, and morphological characteristics support Pyrgulopsis rubra sp. nov. and Pyrgulopsis harrymilleri sp. nov. as distinct from other known Pyrgulopsis species, including the geographically proximate P. metcalfi.
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Manantiales Naturales , Caracoles , Animales , Agua Dulce , TexasRESUMEN
As an approach towards unraveling the nitrogenase mechanism, we have studied the binding of CO to the active-site FeMo-cofactor. CO is not only an inhibitor of nitrogenase, but it is also a substrate, undergoing reduction to hydrocarbons (Fischer-Tropsch-type chemistry). The C-C bond forming capabilities of nitrogenase suggest that multiple CO or CO-derived ligands bind to the active site. Herein, we report a crystal structure with two CO ligands coordinated to the FeMo-cofactor of the molybdenum nitrogenase at 1.33â Å resolution. In addition to the previously observed bridging CO ligand between Fe2 and Fe6 of the FeMo-cofactor, a new ligand binding mode is revealed through a second CO ligand coordinated terminally to Fe6. While the relevance of this state to nitrogenase-catalyzed reactions remains to be established, it highlights the privileged roles for Fe2 and Fe6 in ligand binding, with multiple coordination modes available depending on the ligand and reaction conditions.
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Monóxido de Carbono/metabolismo , Nitrogenasa/metabolismo , Sitios de Unión , Monóxido de Carbono/química , Ligandos , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogenasa/químicaRESUMEN
Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.
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ADP-Ribosilación , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP-Ribosilación/efectos de los fármacos , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Bencimidazoles/farmacología , Línea Celular Tumoral , Técnicas de Visualización de Superficie Celular , Daño del ADN , Glicósido Hidrolasas/metabolismo , Histonas/metabolismo , Humanos , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMEN
There are eight described species in Phreatodrobia, minute, phreatic (subterranean aquatic) snails, all stygobitic and endemic to the Edwards-Trinity Aquifer System of Texas. Two species were described from river drift (Pilsbry Ferriss 1906) and the others more recently by sampling the water flowing from wells or springs (Hershler Longley 1986b; Hershler Longley 1987). Recent sampling from spring orifices and the hyporheic zone of streams have extended the known ranges of the phreatic snails of the region and encountered unknown snails (Alvear et al. 2020). Here we describe Phreatodrobia spica n. sp., a rarely encountered species with a large range of about 400 km (Figure 1). We find P. spica in samples with a diverse assemblage of phreatic animals including other species of Phreatodrobia, isopods, amphipods, coleopterans, and mites. Phreatodrobia spica is distinguished from congeners using morphological and molecular evidence and is characterized by an elevated, trochiform shell with unique sculpture that include spikes and pustules. It has an open umbilicus and a complete, reflected lip that is sometimes appressed to the body whorl.
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Anfípodos , Isópodos , Animales , Ríos , Caracoles , TexasRESUMEN
Protein-protein and protein-ligand interactions often involve conformational changes or structural rearrangements that can be quantified by solution small-angle X-ray scattering (SAXS). These scattering intensity measurements reveal structural details of the bound complex, the number of species involved and, additionally, the strength of interactions if carried out as a titration. Although a core part of structural biology workflows, SAXS-based titrations are not commonly used in drug discovery contexts. This is because prior knowledge of expected sample requirements, throughput and prediction accuracy is needed to develop reliable ligand screens. This study presents the use of the histidine-binding protein (26â kDa) and other periplasmic binding proteins to benchmark ligand screen performance. Sample concentrations and exposure times were varied across multiple screening trials at four beamlines to investigate the accuracy and precision of affinity prediction. The volatility ratio between titrated scattering curves and a common apo reference is found to most reliably capture the extent of structural and population changes. This obviates the need to explicitly model scattering intensities of bound complexes, which can be strongly ligand-dependent. Where the dissociation constant is within 102 of the protein concentration and the total exposure times exceed 20â s, the titration protocol presented at 0.5â mg ml-1 yields affinities comparable to isothermal titration calorimetry measurements. Estimated throughput ranges between 20 and 100 ligand titrations per day at current synchrotron beamlines, with the limiting step imposed by sample handling and cleaning procedures.
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A new species of cave snail (Littorinimorpha: Cochliopidae) in the genus Antrorbis is described from the dark zone of two caves in the Appalachian Valley and Ridge province in eastern Tennessee, United States. The Tennessee Cavesnail, Antrorbis tennesseensis Perez, Shoobs, Gladstone, & Niemiller, sp. nov. is distinguished from its only known congener, Antrorbis breweri, by the absence of raised tubercles on its finely spirally striate protoconch, and its unique radular formula. Moreover, A. tennesseensis is genetically distinct from A. breweri based on substantial divergence at the mitochondrial CO1 locus. This is the first cavesnail to be described from the Appalachian Valley and Ridge (AVR) physiographic province in the state of Tennessee, which previously represented a substantial gap in the distribution of stygobitic (i.e., aquatic, subterranean-obligate) gastropods.
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The ubiquitous and persistent contaminant triclosan is known to cause developmental and behavioral toxicity in fish, but few studies have evaluated the long-term effects of these responses. We used a phenotypically anchored approach to evaluate the behavioral responses caused by early exposure to environmentally relevant concentrations of triclosan to better understand the risk triclosan poses to fish. Zebrafish were exposed to 0, 0.4, 4, or 40 µg triclosan/L (nominal concentrations) for 5 d followed by depuration for 16 d to assess effects on mortality, development, and foraging efficiency. Because foraging efficiency can be impacted by neurological and structural alterations, we assessed morphological and behavioral indicators of neurotoxicity and morphology of craniofacial features associated with gape to identify potential underlying mechanisms associated with altered foraging behaviors. To our knowledge, we are the first to show that early exposure to environmentally relevant concentrations of triclosan impairs foraging efficiency in larval fish by 10%, leading to emaciation and reduced growth and survival. The cause of the impacts of triclosan on foraging efficiency remains unknown, because effects were not associated with overt indicators of neurotoxicity or grossly malformed craniofacial structures. Our results suggest that early exposure to triclosan has the potential to impact the sustainability of wild fish populations, and thus the mechanism underlying behavioral alterations following exposure to triclosan warrants further study. Environ Toxicol Chem 2018;37:3124-3133. © 2018 SETAC.
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Embrión no Mamífero/efectos de los fármacos , Exposición a Riesgos Ambientales , Conducta Alimentaria/efectos de los fármacos , Triclosán/toxicidad , Pez Cebra/embriología , Animales , Encéfalo/anomalías , Encéfalo/efectos de los fármacos , Larva/efectos de los fármacos , Neurotoxinas/toxicidad , Análisis de Supervivencia , Contaminantes Químicos del Agua/toxicidadRESUMEN
Geometric morphometrics allows for the characterization of shape using Cartesian geometric coordinates rather than linear or volumetric measurements, which are dependent upon size and are insufficient to capture geometric shape. By using landmarks on specimens, variations in position, orientation and scale between specimens can be removed to better compare variations in shape. This method has primarily been used in the fields of evolutionary biology and taxonomy. Here we describe how geometric morphometrics can be used to delineate variations in shape caused by teratogenic compounds in zebrafish.
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Estudios de Asociación Genética , Fenotipo , Teratogénesis/genética , Pez Cebra/anomalías , Pez Cebra/genética , Animales , Estudios de Asociación Genética/métodos , Procesamiento de Imagen Asistido por Computador , Microscopía , Programas InformáticosRESUMEN
Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of Praticolella mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding genes.
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Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.
Asunto(s)
Azotobacter vinelandii/enzimología , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Selenio/metabolismo , Azotobacter vinelandii/química , Dominio Catalítico , Cristalografía por Rayos X , Cianatos/metabolismo , Modelos Moleculares , Molibdoferredoxina/química , Conformación Proteica , Compuestos de Selenio/metabolismoRESUMEN
The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO)-inhibited nitrogenase molybdenum-iron (MoFe)-protein at 1.50 angstrom resolution, which reveals a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The µ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 angstrom resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase.