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Batman, the Dark Knight detective
\"In these stories that immediately followed BATMAN: YEAR ONE, the Caped Crusader learns what kind of compromises he must make to be the hero that Gotham City truly needs. As he battles against the deadly Reaper, the city's first vigilante hero, Batman must work with the man who murdered his parents and a cadre of mob bosses to protect Gotham City.\"-- Provided by publisher.
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Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase
A doubly substituted form of the nitrogenase MoFe protein (α-70 ⱽᵃˡ→ᴬˡᵃ, α-195 ᴴⁱˢ→ᴳˡⁿ) has the capacity to catalyze the reduction of carbon dioxide (CO ₂) to yield methane (CH ₄). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH ₄ within 20 min. The catalytic rate depends on the partial pressure of CO ₂ (or concentration of HCO ₃⁻) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H ₂C = CH-CH ₃) through the reductive coupling of CO ₂ and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO ₂ sequestration and formation of olefins.
Journal Article
STRUCTURE, FUNCTION, AND FORMATION OF BIOLOGICAL IRON-SULFUR CLUSTERS
▪ Abstract Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. Owing to their remarkable structural plasticity and versatile chemical/electronic features [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Three different types of [Fe-S] cluster biosynthetic systems have been discovered, and all of them are mechanistically unified by the requirement for a cysteine desulfurase and the participation of an [Fe-S] cluster scaffolding protein. Important mechanistic questions related to [Fe-S] cluster biosynthesis involve the molecular details of how [Fe-S] clusters are assembled on scaffold proteins, how [Fe-S] clusters are transferred from scaffolds to target proteins, how various accessory proteins participate in [Fe-S] protein maturation, and how the biosynthetic process is regulated.
Journal Article
Electron transfer precedes ATP hydrolysis during nitrogenase catalysis
The biological reduction of N ₂ to NH ₃ catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: k ATP = 70 s ⁻¹, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (k ET = 140 s ⁻¹, 25 °C), (ii) ATP hydrolysis (k ATP = 70 s ⁻¹, 25 °C), (iii) Phosphate release (k Pᵢ = 16 s ⁻¹, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (k dᵢₛₛ = 6 s ⁻¹, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe ᵒˣ(ADP) ₂ protein and the reduced MoFe protein.
Journal Article
On reversible H₂ loss upon N₂ binding to FeMo-cofactor of nitrogenase
Nitrogenase is activated for N ₂ reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E ₄, which contains two iron-bridging hydrides [Fe–H–Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H ₂ per N ₂ reduced, which would “waste” two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H ₂ production. In this model, H ₂ is produced by reductive elimination of the two bridging hydrides of E ₄ during N ₂ binding. This process releases H ₂, yielding N ₂ bound to FeMo-cofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D ₂/N ₂, the reverse reaction of D ₂ with the N ₂-bound product of reductive elimination would generate dideutero-E ₄ [E ₄(2D)], which can relax with loss of HD to the state designated E ₂, with a single deuteride bridge [E ₂(D)]. Neither of these deuterated intermediate states could otherwise form in H ₂O buffer. The predicted E ₂(D) and E ₄(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C ₂H ₂) to generate deuterated ethylenes (C ₂H ₃D and C ₂H ₂D ₂). The demonstration that gaseous H ₂/D ₂ can reduce a substrate other than H ⁺ with N ₂ as a cocatalyst confirms the essential mechanistic role for H ₂ formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism.
Journal Article
Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae
Biological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo- R -homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae . NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae . FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.
Journal Article
Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium
Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO₂) to methane (CH₄). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO₂ reduction to CH₄ in vivo using this remodeled nitrogenase. Conversion of CO₂ to CH₄ by R. palustris required constitutive expression of nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH₄ production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO₂ in one enzymatic step using light energy.
Journal Article
Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae
The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.
Journal Article
First‐in‐human safety, tolerability, and pharmacokinetic results of DFV890, an oral low‐molecular‐weight NLRP3 inhibitor
This first‐in‐human study evaluated the safety, tolerability, single‐ and multiple‐dose pharmacokinetic profiles with dietary influence, and pharmacodynamics (PD) of DFV890, an oral NLRP3 inhibitor, in healthy participants. In total, 122 participants were enrolled into a three‐part trial including single and 2‐week multiple ascending oral doses (SAD and MAD, respectively) of DFV890, and were randomized (3:1) to DFV890 or placebo (SAD [3–600 mg] and MAD [fasted: 10–200 mg, once‐daily or fed: 25 and 50 mg, twice‐daily]). DFV890 was generally well‐tolerated. Neither deaths nor serious adverse events were reported. A less than dose‐proportional increase in exposure was observed with the initially used crystalline suspension (3–300 mg); however, an adjusted suspension formulation using spray‐dried dispersion (SDD; 100–600 mg) confirmed dose‐proportional increase in exposure. Relative bioavailability between crystalline suspension and tablets, and food effect were evaluated at 100 mg. Under fasting conditions, Cmax of the tablet yielded 78% compared with the crystalline suspension, and both formulations showed comparable AUC. The fed condition led to a 2.05‐ and 1.49‐fold increase in Cmax and AUC0–last compared with the fasting condition. The median IC50 and IC90 for ex‐vivo lipopolysaccharide‐stimulated interleukin IL‐1β release inhibition (PD) were 61 (90% CI: 50, 70) and 1340 ng/mL (90% CI: 1190, 1490). Crystalline tablets of 100 mg once‐daily or 25 mg twice‐daily were sufficient to maintain ~90% of the IL‐1β release inhibition over 24 h at steady state. Data support dose and formulation selection for further development in diseases, in which an overactivated NLRP3 represents the underlying pathophysiology.
Journal Article