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1. Discovery, isolation, and characterization of diazeniumdiolate siderophores

   https://doi.org/10.1016/bs.mie.2024.06.006  

   Susman, M; Yan, J; Makris, C; Butler, A (July 2024, Elsevier)

   Wencewicz, Timothy (Ed.)

   The C-diazeniumdiolate (N-nitrosohydroxylamine) group in the amino acid graminine (Gra) is a newly discovered Fe(III) ligand in microbial siderophores. Graminine was first identified in the siderophore gramibactin, and since this discovery, other Gra-containing siderophores have been identified, including megapolibactins, plantaribactin, gladiobactin, trinickiabactin (gramibactin B), and tistrellabactins. The C-diazeniumdiolate is photoreactive in UV light which provides a convenient characterization tool for this type of of siderophores. This report details the process of genomics-driven identification of bacteria producing Gra-containing siderophores based on selected biosynthetic enzymes, as well as bacterial culturing, isolation and characterization of the C-diazeniumdiolate siderophores containing Gra. 

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2. Coordination Chemistry of an Emerging Group of Diazeniumdiolate Siderophores: Crystal Structures of M(III)-Gramibactin (M= Fe and Ga)

   https://doi.org/10.1021/jacs.5c13791  

   Thomsen, Emil; Wu, Guang; Butler, Alison (September 2025, Journal of the American Chemical Society)

   To overcome iron starvation, microorganisms often produce siderophores—chelators with high affinity and selectivity for Fe(III). The recent discovery of the siderophore gramibactin garnered significant interest, as it added the C-diazeniumdiolate as a new Fe(III)-binding group in siderophores. Gramibactin is a mixed ligand siderophore, comprised of two graminine residues harboring the diazeniumdiolate donors and a β-hydroxy-aspartate donor. Diazeniumdiolate siderophores have so far evaded crystallographic characterization and few structures of synthetic diazeniumdiolate complexes are reported. To address the gap in structural information, the complexes K[M(III)-gramibactin] (M= Fe and Ga) were prepared, crystallized and their structures solved by X-ray diffraction (XRD). The four Fe-O bond lengths in the two diazeniumdiolates are quite similar, ranging from 1.978 Å to 2.059 Å, indicating an equal contribution in bonding. In contrast, the differing Fe-O bond lengths in β-hydroxy-aspartate reflect the relative donor strengths of the carboxylate (1.997 Å) and alkoxide (1.902 Å) groups. Gramibactin coordinates Fe(III) in a Δ-configured distorted octahedral geometry. The diamagnetic nature of Ga(III) is often leveraged in NMR studies to infer the solution structure of the corresponding Fe(III)-siderophores, which are assumed to be identical. The structural similarity of Ga(III)- and Fe(III)-gramibactin is striking and represents the first crystallographic verification of the assumed isostructural relationship between a Ga(III)- and an Fe(III)-siderophore. By providing concrete evidence, this study promotes Ga(III) as a reliable proxy for Fe(III) in siderophore complexes, with implications for solution structure determination of siderophores and design of Ga(III)-siderophore-based theranostics. 

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3. Stepwise nitrosylation of the nonheme iron site in an engineered azurin and a molecular basis for nitric oxide signaling mediated by nonheme iron proteins

   https://doi.org/10.1039/d1sc00364j  

   Tian, Shiliang; Fan, Ruixi; Albert, Therese; Khade, Rahul L.; Dai, Huiguang; Harnden, Kevin A.; Hosseinzadeh, Parisa; Liu, Jing; Nilges, Mark J.; Zhang, Yong; et al (May 2021, Chemical Science)

   null (Ed.)

   Mononitrosyl and dinitrosyl iron species, such as {FeNO} 7 , {FeNO} 8 and {Fe(NO) 2 } 9 , have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO} 7 and {Fe(NO) 2 } 9 species was achieved, and the elusive {FeNO} 8 species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO} 7 to {Fe(NO) 2 } 9 species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe( ii ) → {FeNO} 7 → {FeNO} 8 → {Fe(NO) 2 } 9 , has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins. 

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4. Stereospecific control of microbial growth by a combinatoric suite of chiral siderophores

   https://doi.org/10.1073/pnas.2423730122  

   Stow, Parker_R; Forsch, Kiefer_O; Thomsen, Emil; Naka, Hiroaki; Haygood, Margo_G; Barbeau, Katherine_A; Butler, Alison (March 2025, Proceedings of the National Academy of Sciences)

   Bacteria compete for iron by producing small-molecule chelators known as siderophores. The triscatechol siderophores trivanchrobactin and ruckerbactin, produced byVibrio campbelliiDS40M4 andYersinia ruckeriYRB, respectively, are naturally occurring diastereomers that form chiral ferric complexes in opposing enantiomeric configurations. Chiral recognition is a hallmark of specificity in biological systems, yet the biological consequences of chiral coordination compounds are relatively unexplored. We demonstrate stereoselective discrimination of microbial growth and iron uptake by chiral Fe(III)–siderophores. The siderophore utilization pathway inV. campbelliiDS40M4 is stereoselective for Λ-Fe(III)–trivanchrobactin, but not the mismatched Δ-Fe(III)–ruckerbactin diastereomer. Chiral recognition is likely conferred by the stereospecificity of both the outer membrane receptor (OMR) protein FvtA and the periplasmic binding protein (PBP) FvtB, both of which must interact preferentially with the Λ-configured Fe(III)-coordination complexes. 

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5. Mining elements of siderophore chirality encoded in microbial genomes

   https://doi.org/10.1002/1873-3468.14539  

   Butler, Alison; Jelowicki, Aneta M.; Ogasawara, Haley A.; Reitz, Zachary L.; Stow, Parker R.; Thomsen, Emil (January 2023, FEBS Letters)

   The vast majority of bacteria require iron to grow. A significant iron acquisition strategy is the production of siderophores, which are secondary microbial metabolites synthesized to sequester iron(III). Siderophore structures encompass a variety of forms, of which highly modified peptidic siderophores are of interest herein. State‐of‐the‐art genome mining tools, such as antiSMASH (antibiotics & Secondary Metabolite Analysis SHell), hold the potential to predict and discover new peptidic siderophores, including a combinatoric suite of triscatechol siderophores framed on a triserine‐ester backbone of the general class, (DHB‐ l / d CAA‐ l Ser) 3 (CAA, cationic amino acid). Siderophores with l / d Arg, l / d Lys and l Orn, but not d Orn, were predicted in bacterial genomes. Fortuitously the d Orn siderophore was identified, yet its lack of prediction highlights the limitation of current genome mining tools. The full combinatoric suite of these siderophores, which form chiral iron(III) complexes, reveals stereospecific coordination chemistry encoded in microbial genomes. The chirality embedded in this suite of Fe(III)‐siderophores raises the question of whether the relevant siderophore‐mediated iron acquisition pathways are stereospecific and selective for ferric siderophore complexes of a defined configuration. 

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