Nitrogenase Cofactor Research Articles

ATP‐Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor

AbstractNitrogenase 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/SO32−‐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 SO32−. 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.

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.

Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5T under conventional and alternative nitrogen fixation

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo‑nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SignificanceIn this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.

Abstract 2516: Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor

Abstract 2516: Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor

Belt-sulfur mobilization in nitrogenase biosynthesis and catalysis

Nitrogenase catalyzes the ambient conversion of N2 and CO to valuable chemical commodities. Recent advances reveal the flexibility of the ‘belt’ region of the nitrogenase cofactor and illustrate the mechanistic importance of belt-sulfur mobilization for the biosynthesis and catalysis of this complex metalloenzyme.

Cover Image

The cover image is based on the Research Article Stability and bonding of carbon(0)-iron N2 complexes relevant to nitrogenase cofactor: EDA-NOCV analyses by Sai Manoj N. V. T. Gorantla et al., https://doi.org/10.1002/jcc.27012

An Fe6 C Core in All Nitrogenase Cofactors.

The biological process of dinitrogen reduction to ammonium occurs at the cofactors of nitrogenases, the only enzymes that catalyze this challenging chemical reaction. Three types of nitrogenases have been described, named according to the heterometal in their cofactor: molybdenum, vanadium or iron nitrogenases. Spectroscopic and structural characterization allowed the unambiguous identification of the cofactors of molybdenum and vanadium nitrogenases and revealed a central μ6 -carbide in both of them. Although genetic studies suggested that the cofactor of the iron nitrogenase contains a similar Fe6 C core, this has not been experimentally demonstrated. Here we report Valence-to-Core X-ray Emission Spectroscopy providing experimental evidence that this cofactor contains a carbide, thereby making the Fe6 C core a feature of all nitrogenase cofactors.

An Fe6C Core in All Nitrogenase Cofactors

AbstractThe biological process of dinitrogen reduction to ammonium occurs at the cofactors of nitrogenases, the only enzymes that catalyze this challenging chemical reaction. Three types of nitrogenases have been described, named according to the heterometal in their cofactor: molybdenum, vanadium or iron nitrogenases. Spectroscopic and structural characterization allowed the unambiguous identification of the cofactors of molybdenum and vanadium nitrogenases and revealed a central μ6‐carbide in both of them. Although genetic studies suggested that the cofactor of the iron nitrogenase contains a similar Fe6C core, this has not been experimentally demonstrated. Here we report Valence‐to‐Core X‐ray Emission Spectroscopy providing experimental evidence that this cofactor contains a carbide, thereby making the Fe6C core a feature of all nitrogenase cofactors.

Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe–9S–C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron–molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe–S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe–4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.

Mackinawite‐Supported Reduction of C1 Substrates into Prebiotically Relevant Precursors

AbstractMackinawite has unique structural properties and reactivities when compared to other iron sulfides. Herein we provide evidence for the mackinawite‐supported reduction of KCN into various reduced compounds under primordial conditions. We proposed a reaction mechanism based on the nucleophilic attack by the deprotonated mackinawite ‐SH surface groups at the carbon atom of HCN. The initial binding of the substrate and the subsequent reduction events are supported by DFT calculations and further experiments using other substrates, such as KSCN, KOCN and CS2. Until now, conversion of CN− into CH4 and NH3 has been limited to nitrogenase cofactors or molecular Fe‐CN complexes. Our study provides evidence for mackinawite‐supported cleavage of the C−N bond under ambient conditions, which opens new avenues for investigation of other substrates for mackinawite‐supported reactions while shedding light on the relevance of this type of reaction to the origin of life on Earth.

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.

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.

Estimations of Fe–N2 Intrinsic Interaction Energies of Iron–Sulfur/Nitrogen–Carbon Sites: A Deeper Bonding Insight by EDA-NOCV Analysis of a Model Complex of the Nitrogenase Cofactor

The MoFe7S9C1– unit of the nitrogenase cofactor (FeMoco) attracts chemists and biochemists due to its unusual ability to bind aerial dinitrogen (N2) at ambient condition and catalytically convert it into ammonia (NH3). The mode of N2 binding and its reaction pathways are yet not clear. An important conclusion has been made based on the very recent synthesis and isolation of model Fe(I/0)-complexes with sulfur-donor ligands under the cleavage of one Fe–S bond followed by binding of N2 at the Fe(0) center. These complexes are structurally relevant to the nitrogenase cofactor (MoFe7S9C1–). Herein, we report the EDA-NOCV analyses and NICS calculations of the dinitrogen-bonded dianionic complex Fe0–N2 (1) (having a CAr ← Fe π-bond) and monoanionic complex FeI–N2 (2) (having a CAr–Fe σ-bond) to provide a deeper insight into the Fe–N2 interacting orbitals and corresponding pairwise interaction energies (EDA-NOCV = energy decomposition analysis coupled with natural orbital for chemical valence; NICS = nucleus-independent chemical shifts). The orbital interaction in the Fe–N2 bond is significantly larger than Coulombic interactions, with major pairwise contributions coming from d(Fe) orbitals to the empty π* orbitals of N2 (three Fe → N2). ΔEint values are in the range of −61 to −77 kcal mol–1. Very interestingly, NICS calculations have been carried out for the fragments before and after binding of the N2 molecule. The computed σ- and π-aromaticity values are attributed to the position of the Fe atoms, oxidation states of Fe centers, and Fe–C bond lengths of these two complexes.

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

The Mo-nitrogenase catalyzes 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 Mo and homocitrate. Previously, we have identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘9th sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘9th sulfur’ insertion remain elusive. Here, we trace the ‘9th sulfur’ insertion using SeO32− and TeO32− as ‘labeled’ SO32−. Biochemical, EPR and XAS/EXAFS studies suggest a role of the ‘9th sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2−- and Te2−-like species into the L-cluster. DFT 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.

Dinitrogen binding and activation at a molybdenum-iron-sulfur cluster.

The Fe-S clusters of nitrogenases carry out the life-sustaining conversion of N2 to NH3. Although progress continues to be made in modelling the structural features of nitrogenase cofactors, no synthetic Fe-S cluster has been shown to form a well-defined coordination complex with N2. Here we report that embedding an [MoFe3S4] cluster in a protective ligand environment enables N2 binding at Fe. The bridging [MoFe3S4]2(μ-η1:η1-N2) complex thus prepared features a substantially weakened N-N bond despite the relatively high formal oxidation states of the metal centres. Substitution of one of the [MoFe3S4] cubanes with an electropositive Ti metalloradical induces additional charge transfer to the N2 ligand with generation of Fe-N multiple-bond character. Structural and spectroscopic analyses demonstrate that N2 activation is accompanied by shortened Fe-S distances and charge transfer from each Fe site, including those not directly bound to N2. These findings indicate that covalent interactions within the cluster play a critical role in N2 binding and activation.

Postbiosynthetic modification of a precursor to the nitrogenase iron–molybdenum cofactor

Significance Biological metalloclusters catalyze many of the global-scale redox interconversions of the elements, such as the nitrogenase-mediated reduction of N 2 to bioavailable NH 3 . Understanding the mechanisms of these transformations requires knowledge of how the clusters’ individual metal ions contribute to their physical properties and chemical reactivity. This has been an ongoing challenge for the Fe–S clusters of nitrogenases, which feature many Fe sites that are difficult to distinguish spectroscopically. Using the L-cluster (a key biosynthetic precursor to the Mo nitrogenase catalytic cofactor) as a case study, we show that chemical modification of nitrogenase cofactors is a promising strategy for simplifying their spectroscopic analysis and relating their electronic structures to their geometric structures.

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

AbstractNifB 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 [Fe4S4] 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.

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.

A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans.

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2 Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivoransIMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.