Structural correlations of nitrogenase active sites using nuclear resonance vibrational spectroscopy and QM/MM calculations.

The biological conversion of N2 to NH3 is accomplished by the nitrogenase family, which is collectively comprised of three closely related but unique metalloenzymes. In the present study, we have employed a combination of the synchrotron-based technique of 57Fe nuclear resonance vibrational spectroscopy together with DFT-based quantum mechanics/molecular mechanics (QM/MM) calculations to probe the electronic structure and dynamics of the catalytic components of each of the three unique M N2ase enzymes (M = Mo, V, Fe) in both the presence (holo-) and absence (apo-) of the catalytic FeMco clusters (FeMoco, FeVco and FeFeco). The results described herein provide vibrational mode assignments for important fingerprint regions of the FeMco clusters, and demonstrate the sensitivity of the calculated partial vibrational density of states (PVDOS) to the geometric and electronic structures of these clusters. Furthermore, we discuss the challenges that are faced when employing NRVS to investigate large, multi-component metalloenzymatic systems, and outline the scope and limitations of current state-of-the-art theory in reproducing complex spectra.

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.

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster.

Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.

Preparation and spectroscopic characterization of lyophilized Mo nitrogenase.

Mo nitrogenase is the primary source of biologically fixed nitrogen, making this system highly interesting for developing new, energy efficient ways of ammonia production. Although heavily investigated, studies of the active site of this enzyme have generally been limited to spectroscopic methods that are compatible with the presence of water and relatively low protein concentrations. One method of overcoming this limitation is through lyophilization, which allows for measurements to be performed on solvent free, high concentration samples. This method also has the potential for allowing efficient protein storage and solvent exchange. To investigate the viability of this preparatory method with Mo nitrogenase, we employ a combination of electron paramagnetic resonance, Mo and Fe K-edge X-ray absorption spectroscopy, and acetylene reduction assays. Our results show that while some small distortions in the metallocofactors occur, oxidation and spin states are maintained through the lyophilization process and that reconstitution of either lyophilized protein component into buffer restores acetylene reducing activity.

The Spectroscopy of Nitrogenases.

Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron-sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

Influenza virus uses transportin 1 for vRNP debundling during cell entry.

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.

Resolving the structure of the E1 state of Mo nitrogenase through Mo and Fe K-edge EXAFS and QM/MM calculations.

Biological nitrogen fixation is predominately accomplished through Mo nitrogenase, which utilizes a complex MoFe7S9C catalytic cluster to reduce N2 to NH3. This cluster requires the accumulation of three to four reducing equivalents prior to binding N2; however, despite decades of research, the intermediate states formed prior to N2 binding are still poorly understood. Herein, we use Mo and Fe K-edge X-ray absorption spectroscopy and QM/MM calculations to investigate the nature of the E1 state, which is formed following the addition of the first reducing equivalent to Mo nitrogenase. By analyzing the extended X-ray absorption fine structure (EXAFS) region, we provide structural insight into the changes that occur in the metal clusters of the protein when forming the E1 state, and use these metrics to assess a variety of possible models of the E1 state. The combination of our experimental and theoretical results supports that formation of E1 involves an Fe-centered reduction combined with the protonation of a belt-sulfide of the cluster. Hence, these results provide critical experiment and computational insight into the mechanism of this important enzyme.

A bound reaction intermediate sheds light on the mechanism of nitrogenase.

Reduction of N2 by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a µ2-bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E6 or E7 of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.

Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase.

Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.

Nitrogenase Cofactor: Inspiration for Model Chemistry.

The cofactor of nitrogenase is the largest and most intricate metal cluster known in nature. Its reactivity, mode of action and even the precise binding site of substrate remain a matter of debate. For decades, synthetic chemists have taken inspiration from the exceptional structural, electronic and catalytic features of the cofactor and have tried to either mimic the unique topology of the entire site, or to extract its functional principles and build them into novel catalysts that achieve the same-or very similar-astounding transformations. We review some of the available model chemistry as it represents the various approaches that have been taken from studying the cofactor, to eventually summarize the current state of knowledge on catalysis by nitrogenase and highlight the mutually beneficial role of model chemistry and enzymology in bioinorganic chemistry.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

Biosynthetic versatility and coordinated action of 5'-deoxyadenosyl radicals in deazaflavin biosynthesis.

Coenzyme F420 is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, F(o)) is still poorly understood. F(o) synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5'-deoxyadenosyl radicals to catalyze F(o) formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a p-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for F(o) synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5'-deoxyadenosyl radicals to mediate the formation of a complex heterocycle.

Biosynthesis of F0, precursor of the F420 cofactor, requires a unique two radical-SAM domain enzyme and tyrosine as substrate.

Cofactors play key roles in metabolic pathways. Among them F(420) has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F(0)-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F(0)-synthase and their in vitro activities. Our study allows us to establish that F(0)-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F(0)-synthase generates two 5'-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.

Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-ß gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-ß expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-ß expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-ß expression and found that PB1-F2-mediated IFN-ß induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.