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 N2 at its surface-exposed L-cluster ([Fe8S9C]), a structural/functional homolog of the M-cluster (or cofactor; [(R-homocitrate)MoFe7S9C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N2 reduction. These observations, coupled with phylogenetic, ecological, and mechanistic considerations, lead to the proposal of a NifEN-like, L-cluster-carrying protein as an ancient nitrogenase, the exploration of which could shed crucial light on the evolutionary origin of nitrogenase and related enzymes.

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.

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.

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. The selectivity of products can be controlled by irradiation conditions, with higher photon flux favoring (longer-chain) hydrocarbon generation. The CZS : VFe biohybrids not only can find applications in industrial CO removal for high-value-added chemical production by using the cheap, renewable solar energy, but also will inspire related research interests in understanding the molecular and electronic processes in photo-biocatalytic systems.

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.

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

The Mo-nitrogenase catalyses the ambient reduction of N2 to NH3 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 N2 via displacement of the belt-sulphur(s) of the M-cluster upon turnover. However, the direct proof of N2 binding and belt-sulphur mobilization during catalysis remains elusive. Here we show that N2 is captured on the M-cluster via electron- and sulphur-depletion, and that the N2-captured state is catalytically competent in generating NH3. Moreover, we demonstrate that product release only occurs when sulphite is supplied along with a reductant, that sulphite is inserted as sulphide into the belt-sulphur displaced positions, and that there is a dynamic in-and-out of the belt-sulphurs during catalysis. Together, these results establish the mobilization of the cofactor belt-sulphurs as a crucial, yet overlooked, mechanistic element of the nitrogenase reaction.

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.

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

Molybdenum nitrogenase catalyses the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe8S8C] 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. Here we trace the 'ninth sulfur' insertion using SeO32- and TeO32- as 'labelled' SO32-. Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the 'ninth sulfur' in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2-- and Te2--like species into the L-cluster. Density functional theory calculations further point to a plausible mechanism involving in situ reduction of SO32- to S2-, thereby suggesting the utility of this reaction to label the catalytically important belt region for mechanistic investigations of nitrogenase.

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.

Response to Comment on "Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase".

Peters et al comment on our report of the dynamic structure of the nitrogenase metallocofactor during N2 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 [Fe4 S4 ] 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. The radical SAM (RS)-cluster is coordinated by three Cys, and the adjacent K1- and K2-clusters, representing the precursor to an 8Fe cofactor core, are each coordinated by one His and two Cys. Prediction of substrate channels, combined with in silico docking of SAM in holo MtNifB, suggests the binding of SAM between the RS- and K2-clusters and putative paths for entry of SAM and exit of products of SAM cleavage, thereby providing important mechanistic insights into the radical SAM-dependent carbide insertion concomitant with cofactor core formation.

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

Nitrogenase converts N2 to NH3 , 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 NifDKCit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDKCit reveals a shift of CO reduction from H2 evolution toward hydrocarbon formation and an opposite shift of N2 reduction from NH3 formation toward H2 evolution. Consistent with a shift in the Mo K-edge energy of NifDKCit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution.

Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase.

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N2) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αß dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo-O5 (carboxyl) or Mo-O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process.

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 [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical.

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 N2. 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. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though key aspects about V- and Fe-nitrogenases remain unexplored.

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.