What Is the Right Level of Activation of a High-Spin {FeNO}7 Complex to Enable Direct N-N Coupling? Mechanistic Insight into Flavodiiron NO Reductases.

Flavodiiron nitric oxide reductases (FNORs), found in pathogenic bacteria, are capable of reducing nitric oxide (NO) to nitrous oxide (N2O) 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, [{FeII(MPA-(RPhO)2)}2] (1, R = H and 2, R = tBu, Me), were prepared first and fully characterized. Complex 1 (without steric protection) directly reduces NO to N2O 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 N2O formation. The implications of these results for FNORs are further discussed.

Distortion of the [FeNO]2 Core in Flavodiiron Nitric Oxide Reductase Models Inhibits N-N Bond Formation and Promotes Formation of Unusual Dinitrosyl Iron Complexes: Implications for Catalysis and Reactivity.

Flavodiiron nitric oxide reductases (FNORs) carry out the reduction of nitric oxide (NO) to nitrous oxide (N2O), allowing infectious pathogens to mitigate toxic levels of NO generated in the human immune response. We previously reported the model complex [Fe2(BPMP)(OPr)(NO)2](OTf)2 (1, OPr- = propionate) that contains two coplanar NO ligands and that is capable of quantitative NO reduction to N2O [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 N2O formation. For this purpose, we prepared several analogues of 1 that contain two monodentate ligands in place of the bridging carboxylate, [Fe2(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 N2O upon one-electron reduction, N2O production is substantially impeded in 2-X, to an initial 5-10% N2O yield. The main products after reduction are unprecedented hs-FeII/{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-FeII/{Fe(NO)2}9/10 DNICs obtained from 2-X show unexpected reactivity and produce up to quantitative N2O yields after 2 h. The implications of these results for the active site structure of FNORs are discussed.

Exploring second coordination sphere effects in flavodiiron nitric oxide reductase model complexes.

Flavodiiron nitric oxide reductases (FNORs) equip pathogens with resistance to nitric oxide (NO), an important immune defense agent in mammals, allowing these pathogens to proliferate in the human body, potentially causing chronic infections. Understanding the mechanism of how FNORs mediate the reduction of NO contributes to the greater goal of developing new therapeutic approaches against drug-resistant strains. Recent density functional theory calculations suggest that a second coordination sphere (SCS) tyrosine residue provides a hydrogen bond that is critical for the reduction of NO to N2O at the active site of FNORs [J. Lu, B. Bi, W. Lai and H. Chen, Origin of Nitric Oxide Reduction Activity in Flavo-Diiron NO Reductase: Key Roles of the Second Coordination Sphere, Angew. Chem., Int. Ed., 2019, 58, 3795-3799]. Specifically, this H-bond stabilizes the hyponitrite intermediate and reduces the energetic barrier for the N-N coupling step. At the same time, the role of the Fe⋯Fe distance and its effect on the N-N coupling step has not been fully investigated. In this study, we equipped the H[BPMP] (= 2,6-bis[[bis(2-pyridylmethyl)amino]methyl]-4-methylphenol) ligand with SCS amide groups and investigated the corresponding diiron complexes with 0-2 bridging acetate ligands. These amide groups can form hydrogen bonds with the bridging acetate ligand(s) and potentially the coordinated NO groups in these model complexes. At the same time, by changing the number of bridging acetate ligands, we can systematically vary the Fe⋯Fe distance. The reactivity of these complexes with NO was then investigated, and the formation of stable iron(II)-NO complexes was observed. Upon one-electron reduction, these NO complexes form Dinitrosyl Iron Complexes (DNICs), which were further characterized using IR and EPR spectroscopy.

Synthesis and characterization of a model complex for flavodiiron NO reductases that stabilizes a diiron mononitrosyl complex.

Flavodiiron NO reductases (FNORs) are important enzymes in microbial pathogenesis, as they equip microbes with resistance to the human immune defense agent nitric oxide (NO). DFT calculations predict that a network of second coordination sphere (SCS) hydrogen bonds is critical for the key NN coupling step in the NO reduction reaction catalyzed by FNORs. In this study, we report the synthesis of a model complex of FNORs with pendant hydrogen bond donors. For this purpose, the ligand H[BPMP] (= 2,6-bis[[bis(2-pyridylmethyl)amino]methyl]-4-methylphenol) was modified with two amide groups in the SCS. Reaction of the precursor complex [Fe2(BPMP(NHCOtBu)2)(OAc)](OTf)2 (1) (OTf- = triflate anion) with NO in the presence of base led to the surprising isolation of a diiron mononitrosyl complex, [Fe2(BPMP(NHCOtBu)(NCOtBu))(OAc)(NO)](OTf) (2) and a triiron decomposition product, [Fe3(BPMP(NHCOtBu)2)(OAc)2(µ-O)2(ONO)](OTf) (3), which were both structurally characterized. Complex 2 models the corresponding mononitrosyl adduct in FNORs. This result points towards a strategy that can be used to stabilize mononitrosyl diiron complexes, using the SCS.