The nitrogenase mechanism: new roles for the dangler?

Dangler sites protruding from a core metallocluster were introduced into the bioinorganic lexicon in 2000 by R.D. Britt and co-workers in an analysis of the tetramanganese oxygen-evolving cluster in photosystem II.

Structural evolution of nitrogenase states under alkaline turnover.

Biological nitrogen fixation, performed by the enzyme nitrogenase, supplies nearly 50% of the bioavailable nitrogen pool on Earth, yet the structural nature of the enzyme intermediates involved in this cycle remains ambiguous. Here we present four high resolution cryoEM structures of the nitrogenase MoFe-protein, sampled along a time course of alkaline reaction mixtures under an acetylene atmosphere. This series of structures reveals a sequence of salient changes including perturbations to the inorganic framework of the FeMo-cofactor; depletion of the homocitrate moiety; diminished density around the S2B belt sulfur of the FeMo-cofactor; rearrangements of cluster-adjacent side chains; and the asymmetric displacement of the FeMo-cofactor.

Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra.

Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach.

Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology.

Nitrogenase beyond the Resting State: A Structural Perspective.

Nitrogenases have the remarkable ability to catalyze the reduction of dinitrogen to ammonia under physiological conditions. How does this happen? The current view of the nitrogenase mechanism focuses on the role of hydrides, the binding of dinitrogen in a reductive elimination process coupled to loss of dihydrogen, and the binding of substrates to a binuclear site on the active site cofactor. This review focuses on recent experimental characterizations of turnover relevant forms of the enzyme determined by cryo-electron microscopy and other approaches, and comparison of these forms to the resting state enzyme and the broader family of iron sulfur clusters.

Modeling the Correlation between and in an X-ray Crystal Structure Refinement.

We have examined how the refined -factor changes as a function of (the atomic number of a scatterer) at the sulfur site of the [4Fe:4S] cluster of the nitrogenase iron protein by refinement. A simple model is developed that quantitatively captures the observed relationship between and , based on a Gaussian electron density distribution with a constant electron density at the position of the scatterer. From this analysis, the fractional changes in and are found to be similar.

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family.

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis.

Structural consequences of turnover-induced homocitrate loss in nitrogenase.

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia during the process of biological nitrogen fixation that is essential for sustaining life. The active site FeMo-cofactor contains a [7Fe:1Mo:9S:1C] metallocluster coordinated with an R-homocitrate (HCA) molecule. Here, we establish through single particle cryoEM and chemical analysis of two forms of the Azotobacter vinelandii MoFe-protein - a high pH turnover inactivated species and a ∆NifV variant that cannot synthesize HCA - that loss of HCA is coupled to α-subunit domain and FeMo-cofactor disordering, and formation of a histidine coordination site.

Solvent Deuterium Isotope Effects of Substrate Reduction by Nitrogenase from .

The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, has been of great interest for understanding the catalytic strategy utilized to reduce dinitrogen to ammonia under ambient temperatures and pressures. The reduction mechanism of nitrogenase is generally envisioned as involving multiple cycles of electron and proton transfers, with the known substrates requiring at least two cycles. Solvent kinetic isotope effect experiments, in which changes of reaction rates or product distribution are measured upon enrichment of solvent with heavy atom isotopes, have been valuable for deciphering the mechanism of complex enzymatic reactions involving proton or hydrogen transfer.

Biological nitrogen fixation in theory, practice, and reality: a perspective on the molybdenum nitrogenase system.

Nitrogenase is the sole enzyme responsible for the ATP-dependent conversion of atmospheric dinitrogen into the bioavailable form of ammonia (NH ), making this protein essential for the maintenance of the nitrogen cycle and thus life itself. Despite the widespread use of the Haber-Bosch process to industrially produce NH , biological nitrogen fixation still accounts for half of the bioavailable nitrogen on Earth. An important feature of nitrogenase is that it operates under physiological conditions, where the equilibrium strongly favours ammonia production.

Computational identification of a systemic antibiotic for gram-negative bacteria.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA.

Selenocyanate derived Se-incorporation into the nitrogenase Fe protein cluster.

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein-independent substrate (CO) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein FeS cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the FeS cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source, an unexpected result as the Fe protein cluster is not traditionally perceived as a site of substrate binding within nitrogenase.

Glutathione binding to the plant Atm3 transporter and implications for the conformational coupling of ABC transporters.

The ATP binding cassette (ABC) transporter of mitochondria (Atm) from (Atm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined four structures of Atm3 in three different conformational states: two inward-facing conformations (with and without bound oxidized glutathione [GSSG]), together with closed and outward-facing states stabilized by MgADP-VO. These structures not only provide a structural framework for defining the alternating access transport cycle, but also reveal the paucity of cysteine residues in the glutathione binding site that could potentially form inhibitory mixed disulfides with GSSG.

Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells.

Modeling the stimulation by glutathione of the steady state kinetics of an adenosine triphosphate binding cassette transporter.

We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the "nonessential activator model" where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function.

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A.

All extant life forms require trace transition metals (e.g., Fe, Cu, and Mn) to survive.

Characterization of the ABC methionine transporter from reveals that lipidated MetQ is required for interaction.

NmMetQ is a substrate-binding protein (SBP) from that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI).

Mechanism of molybdate insertion into pterin-based molybdenum cofactors.

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known.

CaMn O Cubane Models of the Oxygen-Evolving Complex: Spin Ground States S<9/2 and the Effect of Oxo Protonation.

We report the single crystal XRD and MicroED structure, magnetic susceptibility, and EPR data of a series of CaMn O and YMn O complexes as structural and spectroscopic models of the cuboidal subunit of the oxygen-evolving complex (OEC). The effect of changes in heterometal identity, cluster geometry, and bridging oxo protonation on the spin-state structure was investigated. In contrast to previous computational models, we show that the spin ground state of CaMn O complexes and variants with protonated oxo moieties need not be S=9/2.

Microcrystal Electron Diffraction Elucidates Water-Specific Polymorphism-Induced Emission Enhancement of Bis-arylacylhydrazone.

Aggregation-induced emission (AIE) phenomena have gained intense interest over the last decades because of its importance in solid-state emission. However, the elucidation of a working mechanism is difficult owing to the limited characterization methods on solid-state molecules, further complicated if dynamic structural changes occur. Here, a series of bis-arylacylhydrazones () were synthesized, for which their AIE properties are only turned on by the reversible adsorption of water molecules.

Structural Characterization of Two CO Molecules Bound to the Nitrogenase Active Site.

As an approach towards unraveling the nitrogenase mechanism, we have studied the binding of CO to the active-site FeMo-cofactor. CO is not only an inhibitor of nitrogenase, but it is also a substrate, undergoing reduction to hydrocarbons (Fischer-Tropsch-type chemistry). The C-C bond forming capabilities of nitrogenase suggest that multiple CO or CO-derived ligands bind to the active site.

Supplementation with a prebiotic (polydextrose) in obese mouse pregnancy improves maternal glucose homeostasis and protects against offspring obesity.

Objectives: We hypothesised that maternal diet-induced-obesity has adverse consequences for offspring energy expenditure and susceptibility to obesity in adulthood, and that the prebiotic polydextrose (PDX) would prevent the consequences of programming by maternal obesity.

Methods: Female mice were fed a control (Con) or obesogenic diet (Ob) for 6 weeks prior to mating and throughout pregnancy and lactation. Half the obese dams were supplemented with 5% PDX (ObPDX) in drinking water throughout pregnancy and lactation.

Crystal structure of the Escherichia coli transcription termination factor Rho.

During the crystal structure analysis of an ATP-binding cassette (ABC) transporter overexpressed in Escherichia coli, a contaminant protein was crystallized. The identity of the contaminant was revealed by mass spectrometry to be the Escherichia coli transcription terminator factor Rho, structures of which had been previously determined in different conformational states. Although Rho was present at only ∼1% of the target protein (a bacterial homolog of the eukaryotic ABC transporter of mitochondria from Novosphingobium aromaticivorans; NaAtm1), it preferentially crystallized in space group C2 as thin plates that diffracted to 3.

Rethinking the Nitrogenase Mechanism: Activating the Active Site.

Metalloenzymes called nitrogenases (Nases) harness the reactivity of transition metals to reduce N to NH. Specifically, Nases feature a multimetallic active site, called a cofactor, which binds and reduces N. The seven Fe centers and one additional metal center (Mo, V, or Fe) that make up the cofactor are all potential substrate binding sites.