Yisong Guo

Domain of unknown function 4243 (DUF4243) family proteins are an emerging class of nonheme mononuclear iron enzymes characterized by a distinctive iron-binding motif comprised of four histidine (His) and one Nε-carboxylysine (Kcx) residue. Although predominantly found in fungi, OzmD is the only characterized bacterial member. OzmD catalyzes oxidative rearrangement of an aminopyridone ring into a 1,3-oxazine moiety with concomitant cyanide elimination in the final step of oxazinomycin biosynthesis. Herein, the X-ray crystal structure of OzmD is solved, demonstrating the presence of a conserved 4-His-1-Kcx iron-binding motif similar to other DUF4243 family proteins. Biochemical characterization using a substrate analog carrying a hydroxypyridone ring reveals that OzmD also exhibits intrinsic dioxygenase activity, catalyzing a transformation analogous to those of fungal DUF4243 dioxygenases. Electron paramagnetic resonance and Mössbauer spectroscopic characterization of MinCC, a functionally equivalent homologue of OzmD, reveals that the as-isolated protein predominantly contains ferric iron, which can be reduced to the ferrous state upon binding with either the native substrate or the hydroxy analog under anaerobic conditions. These findings support a unified mechanism in which catalysis begins with single-electron transfer from the substrate to ferric iron, enabling molecular oxygen activation and formation of an alkylperoxide intermediate. Overall reaction mechanisms for substrates bearing aminopyridone and hydroxypyridone rings are proposed based on chemical precedents and computational analyses. These findings reveal that OzmD employs a distinctive FeIII-dependent path for oxygen activation in contrast to the classical FeII-dependent route used by most oxygenases and establish a functional connection between bacterial and fungal DUF4243 enzymes.

Synthetic nonheme Fe ^IV ═O complexes reported thus far often have S = 1 ground states, demonstrating markedly lower oxidative reactivity in C─H bond breakage compared to their S = 2 counterparts. Recently, we have identified that incorporating cycloalkyl groups into the second coordination sphere of the nitrilotris(benzimidazole) ligand produce the most reactive Fe ^IV ═O complexes within the tripodal ligand family, exhibiting an S = 1 ground state. Here, we have synthesized six S = 1 complexes having the NTB ligand framework with either electron‐rich or electron‐poor aromatic groups in the secondary coordination sphere of the ligand. These complexes have been characterized by Mössbauer data at 4 K and ¹ H NMR spectra at 193 K with quite similar electronic characteristics despite having distinct electronically tuned ligands. Nevertheless, the complexes show substrate oxidation rates that differ by nearly an order of magnitude from each other, giving rise to a notable linear correlation between activation enthalpies and activation entropies of HAT reactions. These outcomes thus enable further investigation of the Enthalpy‐Entropy Compensation Effect (EECE) in HAT reactions involving these oxoiron(IV) complexes. Finally, the role played by steric attributes of the secondary coordination sphere group in regulating the reactivity of this series of complexes has been explored.

Synthetic nonheme Fe ^IV ═O complexes reported thus far often have S = 1 ground states, demonstrating markedly lower oxidative reactivity in C─H bond breakage compared to their S = 2 counterparts. Recently, we have identified that incorporating cycloalkyl groups into the second coordination sphere of the nitrilotris(benzimidazole) ligand produce the most reactive Fe ^IV ═O complexes within the tripodal ligand family, exhibiting an S = 1 ground state. Here, we have synthesized six S = 1 complexes having the NTB ligand framework with either electron‐rich or electron‐poor aromatic groups in the secondary coordination sphere of the ligand. These complexes have been characterized by Mössbauer data at 4 K and ¹ H NMR spectra at 193 K with quite similar electronic characteristics despite having distinct electronically tuned ligands. Nevertheless, the complexes show substrate oxidation rates that differ by nearly an order of magnitude from each other, giving rise to a notable linear correlation between activation enthalpies and activation entropies of HAT reactions. These outcomes thus enable further investigation of the Enthalpy‐Entropy Compensation Effect (EECE) in HAT reactions involving these oxoiron(IV) complexes. Finally, the role played by steric attributes of the secondary coordination sphere group in regulating the reactivity of this series of complexes has been explored.

Cysteine is a vital amino acid with wide biological utility, serving functions in catalysis, ligand binding, and maintenance of protein structures. In a subset of natural products, termed ribosomally synthesized and post-translationally modified peptides (RiPPs), multiple subclasses are defined by the enzymatic modification of cysteine residues to form thioether bridges. However, RiPPs are short peptides and experimental validation of enzymatically installed thioether crosslinks on large protein substrates is limited. We hypothesized that cysteine-rich proteins whose genes are adjacent to those for enzymes predicted to catalyze RiPP-like modifications might be substrates for such modifications. Thus, we set out to investigate cysteine-rich prokaryotic proteomes for such examples. A bioinformatic analysis identified methanogenic archaea as suitable candidates for further investigation due to the high overall proteome cysteine content, and through targeted proteome scanning, we identified a suitable biosynthetic gene cluster (BGC; Crp) from Methanococcus voltae to characterize. Purification and reconstitution of an annotated radical S-adenosyl-l-methionine (RS) enzyme, CrpC, confirmed that the protein binds three [4Fe-4S] clusters, two of which are in a SPASM domain. Enzyme assays determined that CrpC is active after reconstitution and modifies a cysteine-rich protein substrate in the same BGC, CrpA. Mass spectrometry and chemical derivatization of modified CrpA indicates that CrpC installs ranthionine crosslinks onto CrpA. This research provides a new path for the discovery of intriguing BGCs and post-translational modifications and provides a foundation for the complete structural elucidation and assessment of the native biological function of these modified cysteine-rich proteins.

Sulfur-bridged multimetallic complexes provide a platform for probing long-range electronic communication relevant to metalloproteins and molecular electronic materials. Trimetallic complexes of the type (MN2S2)2Cu (M = [FeNO]2+ or [VO]2+), in which these two redox-active S = 1/2 spin probes are linked by a diamagnetic Cu(I) center in a tetrahedral S4 coordination environment, are developed. Structural analysis shows that the Cu(I) bridge enforces a twisted arrangement of the two MN2S2 units, in contrast to square-planar NiS4-bridged analogues, [ Quiroz, M.; et al., Chem. Sci.2023, 14, 9167–9174]. leading to distinct metal–ligand orbital alignment and sulfur-mediated bonding pathways. Despite a large interspin separation of approximately 6 Å between the spin probes, magnetic susceptibility measurements reveal appreciable ferromagnetic exchange coupling between these paramagnetic centers, although weaker than the coupling observed for the d8 (M′ = Ni(II), Pd(II), and Pt(II))-bridged stair-step complexes. These differences are attributed to the contrasting geometries of the CuS4 and M′S4 cores and their impact on superexchange through the bridging sulfurs. In addition to the solid-state X-ray crystallography, this new class of trimetallics was characterized by multiple methods including electrochemistry, vibrational spectroscopy, magnetic susceptibility, EPR, Mössbauer, and DFT computations. Redox and vibrational studies further probe electronic communication between the FeNO units: ν(NO) stretching frequency indicates that one-electron reduction is largely localized on a single FeNO center, whereas two-electron reduction leads to electron delocalization across both units. Together, these results demonstrate that copper-imposed geometric distortion modulates sulfur-mediated spin coupling and electron delocalization, providing design principles for multimetallic, spin-active architectures.

The field of heme‐catalyzed oxidation chemistry predominantly focuses on oxidations performed by Compounds 0, I, and II. In the past 50 years, a number of alternative high‐valent iron porphyrin species have been identified wherein the porphyrin holds all the oxidative equivalents. Little is known regarding their potential to perform oxidations of small molecules nor their possible role in catalytic pathways. One avenue of particular interest to us is the use of isoporphyrins for the umpolung of chloride ( J. Am. Chem. Soc. 2012 , 134, 4469–4472; Inorg. Chem. 2022 , 61, 8105–8111; Angew. Chem. Int. Ed. 2023 , 62, e202313006). Here we have further explored the viability of such catalytic reactivity and found that chlorination yields remain rather low. One main complication with such reactivity is the presence of a competing pathway. Namely, we are able to efficiently transform 1,3,5‐trimethoxybenzene (TMB) to 2,6‐dimethoxybenzoquinone (DMBQ). The two pathways diverge at the iron(III) π‐dication, as TMB is capable of reducing the π‐dication to a radical π‐cation. We have further investigated the breadth of reactivity of the iron(III) π‐dication and provide comprehensive characterization of the radical π‐cation including UV–vis, resonance Raman, and Mössbauer spectroscopies, as well as mass spectrometry.

The multinuclear nonheme iron-dependent oxidative enzyme (MNIO) family employs a multi-iron cofactor to catalyze a range of post-translational modifications (PTMs) in the biosynthesis of ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. While significant progress has been made toward understanding the range of chemical transformations performed by MNIOs, the nature of the iron cofactor has only been investigated in one instance. Here, we examine the MNIO involved in oxazolin biosynthesis to gain further insight into the metallocofactors employed by this impressive family of enzymes. Oxazolin, a RiPP virulence factor from nontypeable Haemophilus influenzae , contains six copper-binding 5-thiooxazole groups installed by the MNIO HvfB. Weak interactions between HvfB and its required partner protein, HvfC, motivated genetic fusion of the two proteins, which yielded an effective mimic of the protein complex with high enzymatic activity. While HvfB binds up to three iron ions, concerted EPR, ENDOR, and Mössbauer spectroscopic characterization of the active protein reveals that accumulation of a mixed-valent diiron(II/III) cluster correlates with 5-thiooxazole product formation. This oxidation state is attained only in the presence of HvfC, revealing a new role for the partner protein in modulating the iron cofactor. Site-directed mutagenesis of metal-coordinating residues was used to probe the function of the third iron-binding site. This work clarifies the nature of the active iron cofactor for oxazolin maturation, providing a second example of a mixed-valent diiron oxidase in RiPP biosynthesis. TOC Graphic:

Persulfides (RSSH) have been proposed as key players in biochemical transformations that often involve iron, including iron–sulfur cluster assembly, H₂S regulation, post translational modifications, and mitochondrial sulfur oxidation. An example of the latter is found in the O₂-mediated oxidation of glutathione persulfide to sulfite dianion (SO₃^2–) catalyzed by ETHE1, a nonheme iron persulfide dioxygenase (PDO). The iron-mediated mechanism of persulfide oxidation by PDOs remains poorly understood, and there are no synthetic analogues to date. Herein, we report the synthesis, characterization, and O₂ reactivity of a rare iron(II)-alkylpersulfide complex. The adamantyl persulfide anion (AdSS-) was isolated and characterized by X-ray diffraction as a complex with potassium 18-crown-6 [K(18-crown-6)][AdSS], and employed in the synthesis of a new dinuclear iron(II) complex, [(Fe^II(Me₃TACN))₂(μ₂-SSAd)₃][OTf] (1). Complex 1 was characterized by single crystal X-ray diffraction (XRD), UV–vis, ¹H/¹⁹F NMR, and ⁵⁷Fe Mössbauer spectroscopy. Reaction of 1 with O₂ in CH₃CN affords a diiron(III) oxo-bridged complex [(Fe^III(Me₃TACN))₂(μ-O)(μ₂-SO₄)(μ₂-SO₃Ad)][OTf] (2) identified by XRD, and SO₃^2– (∼0.5 equiv per Fe₂). Isotopic labeling studies using ¹⁸O₂ and H₂¹⁸O, supported by control experiments and ESI-MS analysis, indicate that SO₃^2– production proceeds via an iron-centered S-oxygenation mechanism similar to that proposed for persulfide dioxygenases.

We report a novel ¹⁹ F MR imaging probe CoNO2ASF5 exhibiting a large ¹⁹ F chemical shift change of over 30 ppm in response to physiologically relevant pH changes.