ATP-Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor.

Nitrogenase reduces N2 to NH3 at its active-site cofactor. Previous studies of an N2-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP-independent, EuII/SO3 2--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D2-derived D by Av1* into the products of C2H2- and H+-reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO3 2-. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt-sulfur mobilization.

The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach.

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.

The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Organic ligands regulate the environmental impacts of metal-organic frameworks on nitrogen-fixing bacterium Azotobacter vinelandii.

Metal-organic frameworks (MOFs) are rapidly developed materials with fantastic properties and wide applications. The increasing studies highlighted the potential threats of MOF materials to the environment. Comparing to the limited species of metal elements, the organic ligands have much higher diversity, but the influence of organic ligands on the environmental impacts of MOFs has not been revealed. Herein, we synthesized three Cu-MOFs with different organic ligands, namely Cu-BDC (1,4-terephthalic acid), Cu-IM (imidazole) and Cu-TATB (2,4,6-tris(4-carboxyphenyl)- 1,3,5-triazine), and evaluated their environmental toxicity to the nitrogen-fixing bacterium Azotobacter vinelandii. Cu-BDC inhibited the bacterial growth at lower concentrations than Cu-IM and Cu-TATB. The transcriptomes suggested the changes of membrane components by Cu-MOFs, consistent with the membrane leakage and cell wall damages. Cu-MOFs inhibited the nitrogen fixation activity through energy metabolism disturbance according to Gene Ontology functional annotation of ATP binding, Ca2+Mg2+-ATPase activity and ATP content. Only Cu-IM lowered the nitrogen fixation related nif genes, and affected the ribosome, purine metabolism and oxidative phosphorylation pathways. Otherwise, Cu-BDC and Cu-TATB mainly affected the flagellar assemblies and bacterial chemotaxis pathways. Our results collectively indicated that organic ligands regulated the environmental toxicity of MOFs through different metabolism pathways.

Statistical analyses of the oxidized P-clusters in MoFe proteins using the bond-valence method: towards their electron transfer in nitrogenases.

26 well selected oxidized P-clusters (P2+) from the crystallographic data deposited in the Protein Data Bank have been analysed statistically by the bond-valence sum method with weighting schemes for MoFe proteins at different resolutions. Interestingly, the oxidation states of P2+ clusters correspond to Fe23+Fe62+ with high electron delocalization, showing the same oxidation states as the resting states of P-clusters (PN) in nitrogenases. The previously uncertain reduction of P2+ to PN clusters by two electrons was assigned as a double protonation of P2+, in which decoordination of the serine residue and the peptide chain of cysteine take place, in MoFe proteins. This is further supported by the obviously shorter α-alkoxy C-O bond (average of 1.398 Å) in P2+ clusters and longer α-hydroxy C-O bond (average of 1.422 Å) in PN clusters, while no change is observed in the electronic structures of Fe8S7 Fe atoms in P-clusters. Spatially, the calculations show that Fe3 and Fe6, the most oxidized and most reduced Fe atoms, have the shortest distances of 9.329 Šfrom the homocitrate in the FeMo cofactor and 14.947 Šfrom the [Fe4S4] cluster, respectively, and may well function as important electron-transport sites.

Characterization of P(3HB) from untreated raw palm oil mill effluent using Azotobacter vinelandii ΔAvin_16040 lacking S-layer protein.

The production of poly(3-hydroxybutyrate) [P(3HB)] from untreated raw palm oil mill effluent (urPOME), the first wastewater discharge from crude palm oil extraction, is discussed. The mutant strain Azotobacter vinelandii ΔAvin_16040, which lacks the S-layer protein but has a better P(3HB) synthesis capability than the wild type strain ATCC 12,837, was chosen for this study. UrPOME substrate, with high biological oxygen demand (BOD), chemical oxygen demand (COD) and suspended solids, was used without pre-treatment. DSMZ-Azotobacter medium which was devoid of laboratory sugar(s) was used as the basal medium (BaM). Initially, Azotobacter vinelandii ΔAvin_16040 generated 325.5, 1496.3, and 1465.7 mg L-1 of P(3HB) from BaM with 20% urPOME, 2BaM with 20% urPOME and 20 g L-1 sucrose, and 2BaM with 20% urPOME and 2 mL L-1 glycerol, respectively. P(3HB) generation was enhanced by nearly tenfold using statistical optimization, resulting in 13.9 g L-1. Moreover, the optimization reduced the compositions of mineral salts and sugar in the medium by 48 and 97%, respectively. The urPOME-based P(3HB) product developed a yellow coloration most possibly attributed to the aromatic phenolics content in urPOME. Despite the fact that both were synthesised by ΔAvin_16040, thin films of urPOME-based P(3HB) had superior crystallinity and tensile strength than P(3HB) produced only on sucrose. When treated with 10 and 50 kGy of electron beam irradiation, these P(3HB) scissioned to half and one-tenth of their original molecular weights, respectively, and these cleavaged products could serve as useful base units for specific polymer structure construction.

Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration.

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP+ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.

Selenocyanate derived Se-incorporation into the nitrogenase Fe protein cluster.

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein-independent substrate (CO2) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe4S4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe4S4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source, an unexpected result as the Fe protein cluster is not traditionally perceived as a site of substrate binding within nitrogenase. The observed chalcogenide exchange illustrates that this Fe4S4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe protein's repertoire of unique properties.

Many of the molecules that form the building blocks of life contain nitrogen. This element makes up most of the gas in the atmosphere, but in this form, it does not easily react, and most organisms cannot incorporate atmospheric nitrogen into biological molecules. To get around this problem, some species of bacteria produce an enzyme complex called nitrogenase that can transform nitrogen from the air into ammonia. This process is called nitrogen fixation, and it converts nitrogen into a form that can be used to sustain life. The nitrogenase complex is made up of two proteins: the MoFe protein, which contains the active site that binds nitrogen, turning it into ammonia; and the Fe protein, which drives the reaction. Besides the nitrogen fixation reaction, the Fe protein is involved in other biological processes, but it was not thought to bind directly to nitrogen, or to any of the other small molecules that the nitrogenase complex acts on. The Fe protein contains a cluster of iron and sulfur ions that is required to drive the nitrogen fixation reaction, but the role of this cluster in the other reactions performed by the Fe protein remains unclear. To better understand the role of this iron sulfur cluster, Buscagan, Kaiser and Rees used X-ray crystallography, a technique that can determine the structure of molecules. This approach revealed for the first time that when nitrogenase reacts with a small molecule called selenocyanate, the selenium in this molecule can replace the sulfur ions of the iron sulfur cluster in the Fe protein. Buscagan, Kaiser and Rees also demonstrated that the Fe protein could still incorporate selenium ions in the absence of the MoFe protein, which has traditionally been thought to provide the site essential for transforming small molecules. These results indicate that the iron sulfur cluster in the Fe protein may bind directly to small molecules that react with nitrogenase. In the future, these findings could lead to the development of new molecules that artificially produce ammonia from nitrogen, an important process for fertilizer manufacturing. In addition, the iron sulfur cluster found in the Fe protein is also present in many other proteins, so Buscagan, Kaiser and Rees' experiments may shed light on the factors that control other biological reactions.

Crystal structure of the [2Fe-2S] protein I (Shethna protein I) from Azotobacter vinelandii.

Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron-sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Šresolution crystal structure of the [2Fe-2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe-2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe-2S] protein from Aquifex aeolicus, including the positions of the [2Fe-2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins.

Increases in alginate production and transcription levels of alginate lyase (alyA1) by control of the oxygen transfer rate in Azotobacter vinelandii cultures under diazotrophic conditions

BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

Postbiosynthetic modification of a precursor to the nitrogenase iron-molybdenum cofactor.

Nitrogenases utilize Fe-S clusters to reduce N2 to NH3 The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57Fe into specific sites of nitrogenase cofactors, and we report herein site-selective 57Fe labeling of the L-cluster-a carbide-containing, [Fe8S9C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57Fe2+ results in 57Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.

Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii.

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Two-Stage Continuous Conversion of Carbon Monoxide to Ethylene by Whole Cells of Azotobacter vinelandii.

Azotobacter vinelandii is an obligate aerobic diazotroph with a verified transient ability to reduce carbon monoxide to ethylene by its vanadium nitrogenase. In this study, we implemented an industrially relevant continuous two-stage stirred-tank system for in vivo biotransformation of a controlled supply of air enriched with 5% carbon monoxide to 302 µg ethylene g-1 glucose consumed. To attain this value, the process required overcoming critical oxygen limitations during cell proliferation while simultaneously avoiding the A. vinelandii respiratory protection mechanism that negatively impacts in vivo nitrogenase activity. Additionally, process conditions allowed the demonstration of carbon monoxide's solubility as a reaction-limiting factor and a competitor with dinitrogen for the vanadium nitrogenase active site, implying that excess intracellular carbon monoxide could lead to a cessation of cell proliferation and ethylene formation as shown genetically using a new strain of A. vinelandii deficient in carbon monoxide dehydrogenase.IMPORTANCE Ethylene is an essential commodity feedstock used for the generation of a variety of consumer products, but its generation demands energy-intensive processes and is dependent on nonrenewable substrates. This work describes a continuous biological method for investigating the nitrogenase-mediated carbon monoxide reductive coupling involved in ethylene production using whole cells of Azotobacter vinelandii If eventually adopted by industry, this technology has the potential to significantly reduce the total energy input required and the ethylene recovery costs, as well as decreasing greenhouse gas emissions associated with current production strategies.

Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii.

Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.

Development of a bacterial consortium comprising oil-degraders and diazotrophic bacteria for elimination of exogenous nitrogen requirement in bioremediation of diesel-contaminated soil.

The purpose of this study was to develop an effective bacterial consortium and determine their ability to overcome nitrogen limitation for the enhanced remediation of diesel-contaminated soils. Towards this, various bacterial consortia were constructed using oil-degrading and nitrogen-fixing microbes. The diesel removal efficiency of various developed consortia was evaluated by delivering the bacterial consortia to the diesel-contaminated soils. The consortium Acinetobacter sp. K-6 + Rhodococcus sp. Y2-2 + NH4NO3 resulted in the highest removal (85.3%) of diesel from the contaminated soil. The consortium containing two different oil-degrading microbes (K-6 + Y2-2) and one nitrogen-fixing microbe Azotobacter vinelandii KCTC 2426 removed 83.1% of the diesel from the soil after 40 days of treatment. The total nitrogen content analysis revealed higher amounts of nitrogen in soil treated with the nitrogen-fixing microbe when compared with that of the soil supplemented with exogenous inorganic nitrogen. The findings in this present study reveal that the consortium containing the nitrogen-fixing microbe degraded similar amounts of diesel to that degraded by the consortium supplemented with exogenous inorganic nitrogen. This suggests that the developed consortium K-6 + Y2-2 + KCTC 2426 compensated for the nitrogen limitation and eliminated the need for exogenous nitrogen in bioremediation of diesel-contaminated soils.

Nanorg Microbial Factories: Light-Driven Renewable Biochemical Synthesis Using Quantum Dot-Bacteria Nanobiohybrids.

Living cells do not interface naturally with nanoscale materials, although such artificial organisms can have unprecedented multifunctional properties, like wireless activation of enzyme function using electromagnetic stimuli. Realizing such interfacing in a nanobiohybrid organism (or nanorg) requires (1) chemical coupling via affinity binding and self-assembly, (2) the energetic coupling between optoelectronic states of artificial materials with the cellular process, and (3) the design of appropriate interfaces ensuring biocompatibility. Here we show that seven different core-shell quantum dots (QDs), with excitations ranging from ultraviolet to near-infrared energies, couple with targeted enzyme sites in bacteria. When illuminated by light, these QDs drive the renewable production of different biofuels and chemicals using carbon-dioxide (CO2), water, and nitrogen (from air) as substrates. These QDs use their zinc-rich shell facets for affinity attachment to the proteins. Cysteine zwitterion ligands enable uptake through the cell, facilitating cell survival. Together, these nanorgs catalyze light-induced air-water-CO2 reduction with a high turnover number (TON) of ∼106-108 (mols of product per mol of cells) to biofuels like isopropanol (IPA), 2,3-butanediol (BDO), C11-C15 methyl ketones (MKs), and hydrogen (H2); and chemicals such as formic acid (FA), ammonia (NH3), ethylene (C2H4), and degradable bioplastics polyhydroxybutyrate (PHB). Therefore, these resting cells function as nanomicrobial factories powered by light.

The Molybdenum Storage Protein: A soluble ATP hydrolysis-dependent molybdate pump.

A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules ( MoO 4 2 - ) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct ), of only ATP/ADP (MoStobasal ) and of neither ATP/ADP nor POM clusters (MoStozero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 µmolphosphate ·min-1 ·mg-1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg2+ and a weaker occupied ß-ATP/no Mg2+ -binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoStobasal , MoStozero , and MoSto-F1vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4.

Electrocatalytic CO2 reduction catalyzed by nitrogenase MoFe and FeFe proteins.

Nitrogenases catalyze biological dinitrogen (N2) reduction to ammonia (NH3), and also reduce a number of non-physiological substrates, including carbon dioxide (CO2) to formate (HCOO-) and methane (CH4). Three versions of nitrogenase are known (Mo-, V-, and Fe-nitrogenase), each showing different reactivities towards various substrates. Normally, electrons for substrate reduction are delivered by the Fe protein component of nitrogenase, with energy coming from the hydrolysis of 2 ATP to 2 ADP+2 Pi for each electron transferred. Recently, it has been demonstrated that energy and electrons can be delivered from an electrode to the catalytic nitrogenase MoFe-protein without the need for Fe protein or ATP hydrolysis. Here, it is demonstrated that both the MoFe- and FeFe-protein can be immobilized as a polymer layer on an electrode and that electron transfer mediated by cobaltocene can drive CO2 reduction to formate in this system. It was also found that the FeFe-protein diverts a greater percentage of electrons to CO2 reduction versus proton reduction compared to the MoFe-protein. Quantification of electron flow to products exhibited Faradaic efficiencies of CO2 conversion to formate of 9% for MoFe protein and 32% for FeFe-protein, with the remaining electrons going to proton reduction to make H2.

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Previous chemical rescue studies identified a putative FeIII-O2- intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.