Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli.

The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in Escherichia coli.

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe8S7]) and M-cluster core (or L-cluster; [Fe8S9C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.

Enzymatic Fischer-Tropsch-Type Reactions.

The Fischer-Tropsch (FT) process converts a mixture of CO and H2 into liquid hydrocarbons as a major component of the gas-to-liquid technology for the production of synthetic fuels. Contrary to the energy-demanding chemical FT process, the enzymatic FT-type reactions catalyzed by nitrogenase enzymes, their metalloclusters, and synthetic mimics utilize H+ and e- as the reducing equivalents to reduce CO, CO2, and CN- into hydrocarbons under ambient conditions. The C1 chemistry exemplified by these FT-type reactions is underscored by the structural and electronic properties of the nitrogenase-associated metallocenters, and recent studies have pointed to the potential relevance of this reactivity to nitrogenase mechanism, prebiotic chemistry, and biotechnological applications. This review will provide an overview of the features of nitrogenase enzymes and associated metalloclusters, followed by a detailed discussion of the activities of various nitrogenase-derived FT systems and plausible mechanisms of the enzymatic FT reactions, highlighting the versatility of this unique reactivity while providing perspectives onto its mechanistic, evolutionary, and biotechnological implications.

Nitrogenase Fe Protein: A Multi-Tasking Player in Substrate Reduction and Metallocluster Assembly.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO2 or CO to hydrocarbons. This review will provide an overview of the properties and functions of the Fe protein, highlighting the relevance of this unique FeS enzyme to areas related to the catalysis, biosynthesis, and applications of the fascinating nitrogenase system.

Incorporation of an Asymmetric Mo-Fe-S Cluster as an Artificial Cofactor into Nitrogenase.

Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe7 S9 C]), to reduce atmospheric N2 to bioaccessible NH3 . Previously, we have shown that the cofactor-free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe-S cluster [Fe6 S9 (SEt)2 ]4- . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe5 S9 (SH)]3- as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold. The resultant semi-artificial enzyme catalytically reduces C2 H2 to C2 H4 , and CN- into short-chain hydrocarbons, yet it is clearly distinct in activity from its [Fe6 S9 (SEt)2 ]4- -reconstituted counterpart, pointing to the possibility to employ molecular design and cluster synthesis strategies to further develop semi-artificial or artificial systems with desired catalytic activities.

Radical SAM-dependent formation of a nitrogenase cofactor core on NifB.

Nitrogenase is a versatile metalloenzyme that reduces N2, CO and CO2 at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFe7S9C], and it is assembled through the generation of a unique [Fe8S9C] core prior to the insertion of Mo and homocitrate. NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. This review focuses on the recent work that sheds light on the role of NifB in the formation of the [Fe8S9C] core of the nitrogenase cofactor, highlighting the structure, function and mechanism of this unique radical SAM methyltransferase.

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4 Se4 ] Cluster.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe4 S4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe4 Se4 ] cluster that is used to substitute the [Fe4 S4 ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4 Se4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4 Se4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe4 Se4 ]0 ) in the presence of dithionite, where its [Fe4 S4 ] counterpart in AvNifH exists solely in the reduced state ([Fe4 S4 ]1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe4 Se4 ] and [Fe4 S4 ] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen-substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

Probing the All-Ferrous States of Methanogen Nitrogenase Iron Proteins.

The Fe protein of nitrogenase reduces two C1 substrates, CO2 and CO, under ambient conditions when its [Fe4S4] cluster adopts the all-ferrous [Fe4S4]0 state. Here, we show disparate reactivities of the nifH- and vnf-encoded Fe proteins from Methanosarcina acetivorans (designated MaNifH and MaVnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO2 and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that MaVnfH adopts the all-ferrous state at a more positive reduction potential than MaNifH, which could account for the weaker reactivity of the MaVnfH toward C1 substrates than MaNifH. More importantly, MaVnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe4S4]0 cluster at a reduction potential of -0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of -0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe4S4]0 state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe4S4]0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN.

NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli. Biochemical and spectroscopic analyses demonstrate the integrity of the heterologously expressed NifEN in composition and functionality and, additionally, the ability of an engineered NifEN variant to mimic NifDK in retaining the matured cofactor at an analogous cofactor-binding site. This is an important step toward piecing together a viable pathway for the heterologous expression of nitrogenase and identifying variants for the mechanistic investigation of this enzyme.

Crystal structure of 3-[(2-acetamido-phen-yl)imino]-butan-2-one.

In the title compound, 3-[(2-acetamido-phen-yl)imino]-butan-2-one, C12H14N2O2, the imine C=N bond is essentially coplanar with the ketone C=O bond in an s-trans conformation. The benzene ring is twisted away from the plane of the C=N bond by 53.03 (14)°. The acetamido unit is essentially coplanar with the benzene ring. In the crystal, mol-ecules are connected into chains along the c axis through C-H⋯O hydrogen bonds, with two adjacent chains being hinged by C-H⋯O hydrogen bonds.

Transformation of Metal-Organic Framework Secondary Building Units into Hexanuclear Zr-Alkyl Catalysts for Ethylene Polymerization.

We report the stepwise and quantitative transformation of the Zr6(µ3-O)4(µ3-OH)4(HCO2)6 nodes in Zr-BTC (MOF-808) to the [Zr6(µ3-O)4(µ3-OH)4Cl12]6- nodes in ZrCl2-BTC, and then to the organometallic [Zr6(µ3-O)4(µ3-OLi)4R12]6- nodes in ZrR2-BTC (R = CH2SiMe3 or Me). Activation of ZrCl2-BTC with MMAO-12 generates ZrMe-BTC, which is an efficient catalyst for ethylene polymerization. ZrMe-BTC displays unusual electronic and steric properties compared to homogeneous Zr catalysts, possesses multimetallic active sites, and produces high-molecular-weight linear polyethylene. Metal-organic framework nodes can thus be directly transformed into novel single-site solid organometallic catalysts without homogeneous analogs for polymerization reactions.