Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism.

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Aminoquinoline-Based Tridentate (NNN)-Copper Catalyst for C-N Bond-Forming Reactions from Aniline and Diazo Compounds.

A new tridentate Cu2+ complex based on (E)-1-(pyridin-2-yl)-N-(quinolin-8-yl)methanimine (PQM) was generated and characterized to support the activation of diazo compounds for the formation of new C-N bonds. This neutral Schiff base ligand was structurally characterized to coordinate with copper(II) in an equatorial fashion, yielding a distorted octahedral complex. Upon characterization, this copper(II) complex was used to catalyze an efficient and cost-effective protocol for C-N bond formation between N-nucleophiles and copper carbene complexes arising from the activation of diazo carbonyl compounds. A substrate scope of approximately 15 different amine-based substrates was screened, yielding 2° or 3° amine products with acceptable to good yields under mild reaction conditions. Reactivity towards phenol and thiophenol were also screened, showing relatively weak C-O or C-S bond formation under optimized conditions.

Three water-soluble copper(II) N-heterocyclic carbene complexes: toward copper-catalyzed ketone reduction under sustainable conditions.

A series of tridentate copper(II) N-heterocyclic carbene (NHC) complexes with imidazole, benzimidazole, and 5,6-dimethylbenzimidazole azole rings were synthesized and comprehensively characterized via X-ray crystallography, ESI-MS, cyclic voltammetry, and UV-Vis and EPR spectroscopic studies. These complexes were then utilized for the optimization of ketone reduction under sustainable conditions using 2-acetylpyridine and phenylsilane. The relationships between product formation, temperature, reaction time, and catalyst loading for the hydrogenation reactions are covered in detail. Reduction of eighteen different aliphatic, cyclic, and aromatic ketones were demonstrated, which were compatible to produce the corresponding products in moderate to good yields. These systems were used to develop related DNA-hybrid catalytic systems, but only supported weak enantioselectivity. Further thermodynamic experiments showed Cu-NHC complexes did not demonstrate specific binding to DNA, which is consistent with their limited selectivity.

Correction: Magnetic coupling between Fe(NO) spin probe ligands through diamagnetic NiII, PdII and PtII tetrathiolate bridges.

[This corrects the article DOI: 10.1039/D3SC01546G.].

Magnetic coupling between Fe(NO) spin probe ligands through diamagnetic NiII, PdII and PtII tetrathiolate bridges.

Reaction of the nitrosylated-iron metallodithiolate ligand, paramagnetic (NO)Fe(N2S2), with [M(CH3CN)n][BF4]2 salts (M = NiII, PdII, and PtII; n = 4 or 6) affords di-radical tri-metallic complexes in a stairstep type arrangement ([FeMFe]2+, M = Ni, Pd, and Pt), with the central group 10 metal held in a MS4 square plane. These isostructural compounds have nearly identical ν(NO) stretching values, isomer shifts, and electrochemical properties, but vary in their magnetic properties. Despite the intramolecular Fe⋯Fe distances of ca. 6 Å, antiferromagnetic coupling is observed between {Fe(NO)}7 units as established by magnetic susceptibility, EPR, and DFT studies. The superexchange interaction through the thiolate sulfur and central metal atoms is on the order of NiII < PdII ≪ PtII with exchange coupling constants (J) of -3, -23, and -124 cm-1, consistent with increased covalency of the M-S bonds (3d < 4d < 5d). This trend is reproduced by DFT calculations with molecular orbital analysis providing insight into the origin of the enhancement in the exchange interaction. Specifically, the magnitude of the exchange interaction correlates surprisingly well with the energy difference between the HOMO and HOMO-1 orbitals of the triplet states, which is reflected in the central metal's contribution to these orbitals. These results demonstrate the ability of sulfur-dense metallodithiolate ligands to engender strong magnetic communication by virtue of their enhanced covalency and polarizability.

Free ferrous ions sustain activity of mammalian stearoyl-CoA desaturase-1.

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid in a reaction catalyzed by a diiron center. The diiron center is well-coordinated by conserved histidine residues and is thought to remain with the enzyme. However, we find here that SCD1 progressively loses its activity during catalysis and becomes fully inactive after about nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe2+ in cells could regulate SCD1 activity and hence lipid metabolism.

Free ferrous ions sustain activity of mammalian stearoyl-CoA desaturase-1.

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe 2+ ) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe 2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe 2+ in cells could regulate SCD1 activity, and hence lipid metabolism.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

Cooperative redox and spin activity from three redox congeners of sulfur-bridged iron nitrosyl and nickel dithiolene complexes.

The synthesis of sulfur-bridged Fe-Ni heterobimetallics was inspired by Nature's strategies to "trick" abundant first row transition metals into enabling 2-electron processes: redox-active ligands (including pendant iron-sulfur clusters) and proximal metals. Our design to have redox-active ligands on each metal, NO on iron and dithiolene on nickel, resulted in the observation of unexpectedly intricate physical properties. The metallodithiolate, (NO)Fe(N2S2), reacts with a labile ligand derivative of [NiII(S2C2Ph2)]0, NiDT, yielding the expected S-bridged neutral adduct, FeNi, containing a doublet {Fe(NO)}7. Good reversibility of two redox events of FeNi led to isolation of reduced and oxidized congeners. Characterization by various spectroscopies and single-crystal X-ray diffraction concluded that reduction of the FeNi parent yielded [FeNi]-, a rare example of a high-spin {Fe(NO)}8, described as linear FeII(NO-). Mössbauer data is diagnostic for the redox change at the {Fe(NO)}7/8 site. Oxidation of FeNi generated the 2[FeNi]+⇌[Fe2Ni2]2+ equilibrium in solution; crystallization yields only the [Fe2Ni2]2+ dimer, isolated as PF6- and BArF- salts. The monomer is a spin-coupled diradical between {Fe(NO)}7 and NiDT+, while dimerization couples the two NiDT+ via a Ni2S2 rhomb. Magnetic susceptibility studies on the dimer found a singlet ground state with a thermally accessible triplet excited state responsible for the magnetism at 300 K (χMT = 0.67 emu·K·mol-1, µeff = 2.31 µB), and detectable by parallel-mode EPR spectroscopy at 20 to 50 K. A theoretical model built on an H4 chain explains this unexpected low energy triplet state arising from a combination of anti- and ferromagnetic coupling of a four-radical molecular conglomerate.

Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Low-Spin Cyanide Complexes of 3-Mercaptopropionic Acid Dioxygenase (MDO) Reveal the Impact of Outer-Sphere SHY-Motif Residues.

3-Mercaptopropionic acid (3MPA) dioxygenase (MDO) is a non-heme Fe(II)/O2-dependent oxygenase that catalyzes the oxidation of thiol-substrates to yield the corresponding sulfinic acid. Hydrogen-bonding interactions between the Fe-site and a conserved set of three outer-sphere residues (Ser-His-Tyr) play an important catalytic role in the mechanism of this enzyme. Collectively referred to as the SHY-motif, the functional role of these residues remains poorly understood. Here, catalytically inactive Fe(III)-MDO precomplexed with 3MPA was titrated with cyanide to yield a low-spin (S = 1/2) (3MPA/CN)-bound ternary complex (referred to as 1C). UV-visible and electron paramagnetic resonance (EPR) spectroscopy were used to monitor the binding of 3MPA and cyanide. Comparisons of results obtained from SHY-motif variants (H157N and Y159F) were performed to investigate specific H-bonding interactions. For the wild-type enzyme, the binding of 3MPA- and cyanide to the enzymatic Fe-site is selective and results in a homogeneous ternary complex. However, this selectivity is lost for the Y159F variant, suggesting that H-bonding interactions contributed from Tyr159 gate ligand coordination at the Fe-site. Significantly, the g-values for the low-spin ferric site are diagnostic of the directionality of Tyr159 H-bond donation. Computational models coupled with CASSCF/NEVPT2-calculated g-values were used to verify that a major shift in the central g-value (g2) displayed between wild-type and SHY variants could be attributed to the loss of Tyr159 H-bond donation to the Fe-bound cyanide. Applied to native cosubstrate, this H-bond donation provides a means to stabilize Fe-bound dioxygen and potentially explains the attenuated (∼15-fold) rate of catalytic turnover previously reported for MDO SHY-motif variants.

Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid.

To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center.

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

Electron paramagnetic spectrum of dimanganic human serum transferrin.

An EPR signal for Mn(III) bound to the metal transport protein transferrin has been detected for the first time. The temperature dependence and simulations of the EPR signal are consistent with the Mn(III) centers being six-coordinate in an elongated tetragonal environment. Thus, the incorporation of Mn(III) within the Tf active site does not vastly alter the coordination number or active site geometry relative to native Fe(III)2-Tf. This parallel mode EPR signal for Mn(III)2-Tf could prove valuable for future studies aimed at determining the physiological relevance of Mn(III)2-Tf.

Hydrogen Peroxide Disproportionation with Manganese Macrocyclic Complexes of Cyclen and Pyclen.

The catalase family of enzymes, which include a variety with a binuclear manganese active site, mitigate the risk from reactive oxygen species by facilitating the disproportionation of hydrogen peroxide into molecular oxygen and water. In this work, hydrogen peroxide disproportionation using complexes formed between manganese and cyclen or pyclen were investigated due to the spectroscopic similarities with the native MnCAT enzyme. Potentiometric titrations were used to construct speciation diagrams that identify the manganese complex compositions at different pH values. Each complex behaves as a functional mimic of catalase enzymes. UV-visible spectroscopic investigations of the H2O2 decomposition reaction yielded information about the structure of the initial catalyst and intermediates that include monomeric and dimeric species. The results indicate that rigidity imparted by the pyridine ring of pyclen is a key factor in increased TON and TOF values measured compared to cyclen.

1,2-Disubstituted Benzimidazoles by the Iron Catalyzed Cross-Dehydrogenative Coupling of Isomeric o-Phenylenediamine Substrates.

Benzimidazoles are common in nature, medicines, and materials. Numerous strategies for preparing 2-arylbenzimidazoles exist. In this work, 1,2-disubstituted benzimidazoles were prepared from various mono- and disubstituted ortho-phenylenediamines (OPD) by iron-catalyzed oxidative coupling. Specifically, O2 and FeCl3·6H2O catalyzed the cross-dehydrogenative coupling and aromatization of diarylmethyl and dialkyl benzimidazole precursors. N,N'-Disubstituted-OPD substrates were significantly more reactive than their N,N-disubstituted isomers, which appears to be relative to their propensity for complexation and charge transfer with Fe3+. The reaction also converted N-monosubstituted OPD substrates to 2-substituted benzimidazoles; however, electron-poor substrates produce 1,2-disubstituted benzimidazoles by intermolecular imino-transfer. Kinetic, reagent, and spectroscopic (UV-vis and EPR) studies suggest a mechanism involving metal-substrate complexation, charge transfer, and aerobic turnover, involving high-valent Fe(IV) intermediates. Overall, comparative strategies for the relatively sustainable and efficient synthesis of 1,2-disubstituted benzimidazoles are demonstrated.

Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii (Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid (3mpa), l-cysteine (cys), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The pKa values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157-Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa-bound iron-nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.

Catalytic hydrogen atom transfer from hydrosilanes to vinylarenes for hydrosilylation and polymerization.

Because of the importance of hydrogen atom transfer (HAT) in biology and chemistry, there is increased interest in new strategies to perform HAT in a sustainable manner. Here, we describe a sustainable, net redox-neutral HAT process involving hydrosilanes and alkali metal Lewis base catalysts - eliminating the use of transition metal catalysts - and report an associated mechanism concerning Lewis base-catalysed, complexation-induced HAT (LBCI-HAT). The catalytic LBCI-HAT is capable of accessing both branch-specific hydrosilylation and polymerization of vinylarenes in a highly selective fashion, depending on the Lewis base catalyst used. In this process, earth abundant, alkali metal Lewis base catalyst plays a dual role. It first serves as a HAT initiator and subsequently functions as a silyl radical stabilizing group, which is critical to highly selective cross-radical coupling. EPR study identified a potassiated paramagnetic species and multistate density function theory revealed a high HAT character, yet multiconfigurational nature in the transition state of the reaction.

Isoindolinone Synthesis: Selective Dioxane-Mediated Aerobic Oxidation of Isoindolines.

N-Alkyl and N-aryl-isoindolinones were prepared by a dioxane-mediated oxidation of isoindoline precursors. The transformation exhibits unique chemoselectivity for isoindonlines. A chiral tertiary (3°)-benzylic position was not racemized during oxidation, and methyl indoprofen was prepared by late stage oxidation. Mechanistic studies suggest a selective H atom transfer, which avoids many known oxidation (by-)products of isoindolinones.

Structural and Electronic Responses to the Three Redox Levels of Fe(NO)N2 S2 -Fe(NO)2.

The nitrosylated diiron complexes, Fe2 (NO)3 , of this study are interpreted as a mono-nitrosyl Fe(NO) unit, MNIU, within an N2 S2 ligand field that serves as a metallodithiolate ligand to a dinitrosyl iron unit, DNIU. The cationic Fe(NO)N2 S2 ⋅Fe(NO)2 + complex, 1+ , of Enemark-Feltham electronic notation {Fe(NO)}7 -{Fe(NO)2 }9 , is readily obtained via myriad synthetic routes, and shown to be spin coupled and diamagnetic. Its singly and doubly reduced forms, {Fe(NO)}7 -{Fe(NO)2 }10 , 10 , and {Fe(NO)}8 -{Fe(NO)2 }10 , 1- , were isolated and characterized. While structural parameters of the DNIU are largely unaffected by redox levels, the MNIU readily responds; the neutral, S= 1 / 2 , complex, 10 , finds the extra electron density added into the DNIU affects the adjacent MNIU as seen by the decrease its Fe-N-O angle (from 171° to 149°). In contrast, addition of the second electron, now into the MNIU, returns the Fe-N-O angle to 171° in 1- . Compensating shifts in FeMNIU distances from the N2 S2 plane (from 0.518 to 0.551 to 0.851 Å) contribute to the stability of the bimetallic complex. These features are addressed by computational studies which indicate that the MNIU in 1- is a triplet-state {Fe(NO)}8 with strong spin polarization in the more linear FeNO unit. Magnetic susceptibility and parallel mode EPR results are consistent with the triplet state assignment.