Investigating the Molybdenum Nitrogenase Mechanistic Cycle Using Spectroelectrochemistry.

Molybdenum nitrogenase plays a crucial role in the biological nitrogen cycle by catalyzing the reduction of dinitrogen (N) to ammonia (NH) under ambient conditions. However, the underlying mechanisms of nitrogenase catalysis, including electron and proton transfer dynamics, remain only partially understood. In this study, we covalently attached molybdenum nitrogenase (MoFe) to gold electrodes and utilized surface-enhanced infrared absorption spectroscopy (SEIRA) coupled with electrochemistry techniques to investigate its catalytic mechanism.

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

The biological conversion of N to NH 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 Fe 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 Nase 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.

An Fe 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 μ -carbide in both of them.

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 NifDK polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifDK. P-cluster maturation involves the iron protein NifH as well as several accessory proteins, whose role has not been elucidated.

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.

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 N to 2NH. 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.

Resolving the structure of the E 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 MoFeSC catalytic cluster to reduce N to NH. This cluster requires the accumulation of three to four reducing equivalents prior to binding N; however, despite decades of research, the intermediate states formed prior to N 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 E state, which is formed following the addition of the first reducing equivalent to Mo nitrogenase.

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.

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

Reduction of N 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.

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

Radical -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 C 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 , and a reaction mechanism has been proposed.

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.

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 N 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.

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.

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.

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.