Ammonia synthesis via an engineered nitrogenase assembly pathway in .

Heterologous expression of nitrogenase has been actively pursued because of the far-reaching impact of this enzyme on agriculture, energy and environment. Yet, isolation of an active two-component, metallocentre-containing nitrogenase from a non-diazotrophic host has yet to be accomplished. Here, we report the heterologous synthesis of an active Mo-nitrogenase by combining genes from and in .

NifEN: a versatile player in nitrogenase assembly, catalysis and evolution.

The Mo-nitrogenase catalyzes the reduction of N to NH at the cofactor of its catalytic NifDK component. NifEN shares considerable homology with NifDK in primary sequence, tertiary structure and associated metallocenters. Better known for its biosynthetic function to convert an all-iron precursor (L-cluster; [FeSC]) to a mature cofactor (M-cluster; [(R-homocitrate) MoFeSC]), NifEN also mimics NifDK in catalyzing substrate reduction at ambient conditions.

Cofactor maturase NifEN: A prototype ancient nitrogenase?

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N at its surface-exposed L-cluster ([FeSC]), a structural/functional homolog of the M-cluster (or cofactor; [(-homocitrate)MoFeSC]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N reduction.

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

Nitrogenase reduces N to NH at its active-site cofactor. Previous studies of an N-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N species on Av1* remains elusive. Here we report a study of ATP-independent, Eu/SO -driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques.

Heterologous expression of a fully active nitrogenase Fe protein in .

The heterologous expression of a fully active 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 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 .

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 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 ([FeS]) and M-cluster core (or L-cluster; [FeSC]) 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 , , , and genes, and that of an L-cluster-containing NifB protein upon coexpression of , and genes alongside the gene, in .

Light-driven Transformation of Carbon Monoxide into Hydrocarbons using CdS@ZnS : VFe Protein Biohybrids.

Enzymatic Fisher-Tropsch (FT) process catalyzed by vanadium (V)-nitrogenase can convert carbon monoxide (CO) to longer-chain hydrocarbons (>C2) under ambient conditions, although this process requires high-cost reducing agent(s) and/or the ATP-dependent reductase as electron and energy sources. Using visible light-activated CdS@ZnS (CZS) core-shell quantum dots (QDs) as alternative reducing equivalent for the catalytic component (VFe protein) of V-nitrogenase, we first report a CZS : VFe biohybrid system that enables effective photo-enzymatic C-C coupling reactions, hydrogenating CO into hydrocarbon fuels (up to C4) that can be hardly achieved with conventional inorganic photocatalysts. Surface ligand engineering optimizes molecular and opto-electronic coupling between QDs and the VFe protein, realizing high efficiency (internal quantum yield >56 %), ATP-independent, photon-to-fuel production, achieving an electron turnover number of >900, that is 72 % compared to the natural ATP-coupled transformation of CO into hydrocarbons by V-nitrogenase.

Enzymatic Fischer-Tropsch-Type Reactions.

The Fischer-Tropsch (FT) process converts a mixture of CO and H 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, CO, and CN into hydrocarbons under ambient conditions. The C 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.

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 CO or CO to hydrocarbons.

Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor.

The Mo-nitrogenase catalyses the ambient reduction of N to NH at the M-cluster, a complex cofactor that comprises two metal-sulphur partial cubanes ligated by an interstitial carbide and three belt-sulphurs. A recent crystallographic study suggests binding of N via displacement of the belt-sulphur(s) of the M-cluster upon turnover. However, the direct proof of N binding and belt-sulphur mobilization during catalysis remains elusive.

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)MoFe S C]), to reduce atmospheric N to bioaccessible NH . 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 [Fe S (SEt) ] . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe S (SH)] as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold.

Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase.

Gases like H, N, CO, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N, CO, and CO and the production of H require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur.

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

Nitrogenase is a versatile metalloenzyme that reduces N, CO and CO at its cofactor site. Designated the M-cluster, this complex cofactor has a composition of [(R-homocitrate)MoFeSC], and it is assembled through the generation of a unique [FeSC] 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.

Tracing the incorporation of the "ninth sulfur" into the nitrogenase cofactor precursor with selenite and tellurite.

Molybdenum nitrogenase catalyses the reduction of N to NH at its cofactor, an [(R-homocitrate)MoFeSC] cluster synthesized via the formation of a [FeSC] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [FeSC] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the 'ninth sulfur' in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon 'ninth sulfur' insertion remain elusive.

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

The Fe protein of nitrogenase reduces two C1 substrates, CO and CO, under ambient conditions when its [FeS] cluster adopts the all-ferrous [FeS] state. Here, we show disparate reactivities of the - and -encoded Fe proteins from (designated NifH and VnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO 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 VnfH adopts the all-ferrous state at a more positive reduction potential than NifH, which could account for the weaker reactivity of the VnfH toward C1 substrates than NifH.

An EPR and VTVH MCD spectroscopic investigation of the nitrogenase assembly protein NifB.

NifB, a radical SAM enzyme, catalyzes the biosynthesis of the L cluster (FeSC), a structural homolog and precursor to the nitrogenase active-site M cluster ([MoFeSC·R-homocitrate]). Sequence analysis shows that NifB contains the CxxCxxxC motif that is typically associated with the radical SAM cluster ([FeS]) involved in the binding of S-adenosylmethionine (SAM). In addition, NifB houses two transient [FeS] clusters (K cluster) that can be fused into an 8Fe L cluster concomitant with the incorporation of an interstitial carbide ion, which is achieved through radical SAM chemistry initiated at the [FeS] cluster upon its interaction with SAM.

Response to Comment on "Structural evidence for a dynamic metallocofactor during N reduction by Mo-nitrogenase".

Peters comment on our report of the dynamic structure of the nitrogenase metallocofactor during N reduction. Their claim that their independent structural refinement and consideration of biochemical data contradict our finding is incorrect and is strongly refuted by our biochemical and structural data that collectively and conclusively point to the binding of dinitrogen species to the nitrogenase cofactor.

X-Ray Crystallographic Analysis of NifB with a Full Complement of Clusters: Structural Insights into the Radical SAM-Dependent Carbide Insertion During Nitrogenase Cofactor Assembly.

NifB is an essential radical SAM enzyme required for the assembly of an 8Fe core of the nitrogenase cofactor. Herein, we report the X-ray crystal structures of Methanobacterium thermoautotrophicum NifB without (apo MtNifB) and with (holo MtNifB) a full complement of three [Fe S ] clusters. Both apo and holo MtNifB contain a partial TIM barrel core, but unlike apo MtNifB, holo MtNifB is fully assembled and competent in cofactor biosynthesis.

Characterization of a Mo-Nitrogenase Variant Containing a Citrate-Substituted Cofactor.

Nitrogenase converts N to NH , and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDK , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue.

Structural evidence for a dynamic metallocofactor during N reduction by Mo-nitrogenase.

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.

Special Issue on Nitrogenases and Homologous Systems.

The nitrogenase superfamily comprises homologous enzyme systems that carry out fundamentally important processes, including the reduction of N and CO, and the biosynthesis of bacteriochlorophyll and coenzyme F430. This special issue provides a cross-disciplinary overview of the ongoing research in this highly diverse and unique research area of metalloprotein biochemistry.

Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB.

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [FeS] clusters (designated K1 and K2) concomitant with carbide insertion into an [FeSC] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive.

Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases.

Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N. Nitrogenase is most commonly associated with the molybdenum-iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two "alternative nitrogenase" systems were discovered, isolated, and found to incorporate V or Fe in place of Mo.