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High-valent nonheme FeIV-oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal-oxido complexes. To address this knowledge gap, we introduced a series of FeIV-oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat]3-, which contains phosphinic amido groups. An important structural aspect of [FeIVpoat(O)]- is the inclusion of an auxiliary site capable of binding a Lewis acid (LAII); we used this unique feature to further modulate the electrostatic environment around the Fe-oxido unit. Experimentally, studies confirmed that H-bonds and LAII s can interact directly with the oxido ligand in FeIV-oxido complexes, which weakens the Fe═O bond and has an impact on the electronic structure. We found that relatively large vibrational changes in the Fe-oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.

Flavodiiron nitric oxide reductases (FNORs), found in pathogenic bacteria, are capable of reducing nitric oxide (NO) to nitrous oxide (N 2 O) to detoxify NO released by the human immune system. Previously, we reported the first FNOR model system that mediates direct NO reduction (Dong, H. T.; J. Am. Chem. Soc. 2018, 140, 13429-13440), but no intermediate of the reaction could be characterized. Here, we present a new set of model complexes that, depending on the ligand substitution, can either mediate direct NO reduction or stabilize a highly activated high-spin (hs) {FeNO} 7 complex, the first intermediate of the reaction. The precursors, [{Fe II (MPA-(RPhO) 2 )} 2 ] ( 1 , R = H and 2 , R = t Bu, Me), were prepared first and fully characterized. Complex 1 (without steric protection) directly reduces NO to N 2 O almost quantitatively, which constitutes only the second example of this reaction in model systems. Contrarily, the reaction of sterically protected 2 with NO forms the stable mononitrosyl complex 3 , which shows one of the lowest N-O stretching frequencies (1689 cm -1 ) observed so far for a mononuclear hs-{FeNO} 7 complex. This study confirms that an N-O stretch ≤1700 cm -1 represents the appropriate level of activation of the FeNO unit to enable direct NO reduction. The higher activation level of these hs-{FeNO} 7 complexes required for NO reduction compared to those formed in FNORs emphasizes the importance of hydrogen bonding residues in the active sites of FNORs to activate the bound NO ligands for direct N-N coupling and N 2 O formation. The implications of these results for FNORs are further discussed.

Flavodiiron nitric oxide reductases (FNORs) carry out the reduction of nitric oxide (NO) to nitrous oxide (N 2 O), allowing infectious pathogens to mitigate toxic levels of NO generated in the human immune response. We previously reported the model complex [Fe 2 (BPMP)(OPr)(NO) 2 ](OTf) 2 ( 1 , OPr - = propionate) that contains two coplanar NO ligands and that is capable of quantitative NO reduction to N 2 O [White et al. J. Am. Chem. Soc. 2018 , 140 , 2562-2574]. Here we investigate, for the first time, how a distortion of the active site affects the ability of the diiron core to mediate N 2 O formation. For this purpose, we prepared several analogues of 1 that contain two monodentate ligands in place of the bridging carboxylate, [Fe 2 (BPMP)(X) 2 (NO) 2 ] 3+/1+ ( 2-X ; X = triflate, 1-methylimidazole, or methanol). Structural data of 2-X show that without the bridging carboxylate, the diiron core expands, leading to elongated (O)N-N(O) distances (from 2.80 Å in 1 to 3.00-3.96 Å in 2-X ) and distorted (O)N-Fe-Fe-N(O) dihedral angles (from coplanarity (5.9°) in 1 to 52.9-85.1° in 2-X ). Whereas 1 produces quantitative amounts of N 2 O upon one-electron reduction, N 2 O production is substantially impeded in 2-X , to an initial 5-10% N 2 O yield. The main products after reduction are unprecedented hs-Fe II /{Fe(NO) 2 } 9/10 dinitrosyl iron complexes (DNICs). Even though mononuclear DNICs are stable and do not show N-N coupling (since it is a spin-forbidden process), the hs-Fe II /{Fe(NO) 2 } 9/10 DNICs obtained from 2-X show unexpected reactivity and produce up to quantitative N 2 O yields after 2 h. The implications of these results for the active site structure of FNORs are discussed.

The extradiol dioxygenases are a large subclass of mononuclear nonheme Fe enzymes that catalyze the oxidative cleavage of catechols distal to their OH groups. These enzymes are important in bioremediation, and there has been significant interest in understanding how they activate O 2 . The extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) provides an opportunity to study this process, as two O 2 intermediates have been trapped and crystallographically defined using the slow substrate 4-nitrocatechol (4NC): a side-on Fe-O 2 -4NC species and a Fe-O 2 -4NC peroxy bridged species. Also with 4NC, two solution intermediates have been trapped in the H200N variant, where H200 provides a second-sphere hydrogen bond in the wild-type enzyme. While the electronic structure of these solution intermediates has been defined previously as Fe III -superoxo-catecholate and Fe III -peroxy-semiquinone, their geometric structures are unknown. Nuclear resonance vibrational spectroscopy (NRVS) is an important tool for structural definition of nonheme Fe-O 2 intermediates, as all normal modes with Fe displacement have intensity in the NRVS spectrum. In this study, NRVS is used to define the geometric structure of the H200N-4NC solution intermediates in HPCD as an end-on Fe III -superoxo-catecholate and an end-on Fe III -hydroperoxo-semiquinone. Parallel calculations are performed to define the electronic structures and protonation states of the crystallographically defined wild-type HPCD-4NC intermediates, where the side-on intermediate is found to be a Fe III -hydroperoxo-semiquinone. The assignment of this crystallographic intermediate is validated by correlation to the NRVS data through computational removal of H200. While the side-on hydroperoxo semiquinone intermediate is computationally found to be nonreactive in peroxide bridge formation, it is isoenergetic with a superoxo catecholate species that is competent in performing this reaction. This study provides insight into the relative reactivities of Fe III -superoxo and Fe III -hydroperoxo intermediates in nonheme Fe enzymes and into the role H200 plays in facilitating extradiol catalysis.

Heme and non-heme iron-nitrosyl complexes are important intermediates in biology. While there are numerous examples of low-spin heme iron-nitrosyl complexes in different oxidation states, much less is known about high-spin (hs) non-heme iron-nitrosyls in oxidation states other than the formally ferrous NO adducts ({FeNO} 7 in the Enemark-Feltham notation). In this study, we present a complete series of hs-{FeNO} 6-8 complexes using the TMG 3 tren coligand. Redox transformations from the hs-{FeNO} 7 complex [Fe(TMG 3 tren)(NO)] 2+ to its {FeNO} 6 and {FeNO} 8 analogs do not alter the coordination environment of the iron center, allowing for detailed comparisons between these species. Here, we present new MCD, NRVS, XANES/EXAFS, and Mössbauer data, demonstrating that these redox transformations are metal based, which allows us to access hs-Fe(II)-NO - , Fe(III)-NO - , and Fe(IV)-NO - complexes. Vibrational data, analyzed by NCA, directly quantify changes in Fe-NO bonding along this series. Optical data allow for the identification of a "spectator" charge-transfer transition that, together with Mössbauer and XAS data, directly monitors the electronic changes of the Fe center. Using EXAFS, we are also able to provide structural data for all complexes. The magnetic properties of the complexes are further analyzed (from magnetic Mössbauer). The properties of our hs-{FeNO} 6-8 complexes are then contrasted to corresponding, low-spin iron-nitrosyl complexes where redox transformations are generally NO centered. The hs-{FeNO} 8 complex can further be protonated by weak acids, and the product of this reaction is characterized. Taken together, these results provide unprecedented insight into the properties of biologically relevant non-heme iron-nitrosyl complexes in three relevant oxidation states.

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin Fe III -(hydro)peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native Fe II /O 2 reaction is evaluated. For the native Fe II /O 2 reaction, an Fe III -superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of Fe III -superoxo and Fe III -hydroperoxo species in nonheme Fe biochemistry.

Nitrogenase catalyzes a reaction critical for life, the reduction of N(2) to 2NH(3), yet we still know relatively little about its catalytic mechanism. We have used the synchrotron technique of (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the dynamics of the Fe-S clusters in this enzyme. The catalytic site FeMo-cofactor exhibits a strong signal near 190 cm(-)(1), where conventional Fe-S clusters have weak NRVS. This intensity is ascribed to cluster breathing modes whose frequency is raised by an interstitial atom. A variety of Fe-S stretching modes are also observed between 250 and 400 cm(-)(1). This work is the first spectroscopic information about the vibrational modes of the intact nitrogenase FeMo-cofactor and P-cluster.