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

© 2021 National Academy of Sciences. All rights reserved. Nitrogenases utilize Fe-S clusters to reduce N2to NH3. The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57Fe into specific sites of nitrogenase cofactors, and we report herein siteselective 57Fe labeling of the L-cluster-a carbide-containing, [Fe8S9C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57Fe2+results in 57Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecularlevel insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors

Connecting the Geometric and Electronic Structures of the Nitrogenase Iron–Molybdenum Cofactor through Site-selective Labeling

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle in these efforts has been that the 57Fe-based spectroscopic data—although rich with information—reflects all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the 3-D structure. We herein overcome this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the reduced and oxidized forms of the resting state provides unprecedented insights into the local electronic structure of the terminal (Fe1) site, including its oxidation state and spin orientation. This leads to the discovery that Fe1 is a site of redox reactivity during oxidation of the resting state, and on this basis, we suggest a possible role for Fe1 as an electron reservoir during N2 reduction catalysis

NosN, a Radical <i>S</i>‑Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide

Nosiheptide, a member of the <i>e</i> series of macrocyclic thiopeptide natural products, contains a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide backbone via ester and thioester linkages, respectively. Herein, we show that NosN, a predicted class C radical <i>S</i>-adenosylmethionine (SAM) methylase, catalyzes both the transfer of a C1 unit from SAM to 3-methylindolic acid linked to Cys8 of a synthetic substrate surrogate as well as the formation of the ester linkage between Glu6 and the nascent C4 methylene moiety of DMIA. In contrast to previous studies that indicated that 5′-methylthioadenosine is the immediate methyl donor in the reaction, in our studies, SAM itself plays this role, giving rise to <i>S</i>-adenosylhomocysteine as a coproduct of the reaction

Evaluation of the effect of valence state on cerium oxide nanoparticle toxicity following intratracheal instillation in rats

<p>Cerium (Ce) is becoming a popular metal for use in electrochemical applications. When in the form of cerium oxide (CeO₂), Ce can exist in both 3 + and 4 + valence states, acting as an ideal catalyst. Previous <i>in vitro</i> and <i>in vivo</i> evidence have demonstrated that CeO₂ has either anti- or pro-oxidant properties, possibly due to the ability of the nanoparticles to transition between valence states. Therefore, we chose to chemically modify the nanoparticles to shift the valence state toward 3+. During the hydrothermal synthesis process, 10 mol% gadolinium (Gd) and 20 mol% Gd, were substituted into the lattice of the CeO₂ nanoparticles forming a perfect solid solution with various A-site valence states. These two Gd-doped CeO₂ nanoparticles were compared to pure CeO₂ nanoparticles. Preliminary characteristics indicated that doping results in minimal size and zeta potential changes but alters valence state. Following characterization, male Sprague-Dawley rats were exposed to 0.5 or 1.0 mg/kg nanoparticles via a single intratracheal instillation. Animals were sacrificed and bronchoalveolar lavage fluid and various tissues were collected to determine the effect of valence state and oxygen vacancies on toxicity 1-, 7-, or 84-day post-exposure. Results indicate that damage, as measured by elevations in lactate dehydrogenase, occurred within 1-day post-exposure and was sustained 7-day post-exposure, but subsided to control levels 84-day post-exposure. Furthermore, no inflammatory signaling or lipid peroxidation occurred following exposure with any of the nanoparticles. Our results implicate that valence state has a minimal effect on CeO₂ nanoparticle toxicity <i>in vivo</i>.</p