Modelling Flavodiiron Nitric Oxide Reductases: Geometric and Electronic Structure of Key Intermediates and Mechanistic Insights

Abstract

In the face of increasing bacterial resistance to known antibiotics, there is an increasing motivation to study the routes by which bacteria can proliferate. One such route involves the bacterial use of flavodiiron nitric oxide reductases (FNORs), a subclass of flavodiiron proteins (FDPs), to carry out the reduction of nitric oxide (NO) to nitrous oxide (N2O), thus mitigating the accumulation of toxic levels of NO generated in the human immune response. While enzyme studies on the Thermatoga maritima FDP detailed a preliminary route where a high-spin diiron dinitrosyl complex (hs-[{Fe(NO)}7]2 in Enemark-Feltham notation) accumulates before releasing N2O in the presence or absence of the enzyme’s flavin (FMN) cofactor, the structural and electronic prerequisites for non-heme diiron cores to efficiently carry out this reaction had not been systematically studied prior to this work. Here, several hs-[{Fe(NO)}7]2 and hs-FeII{Fe(NO)}7 model complexes were synthesized and their mechanisms of NO reduction were characterized to identify (1) the electronic prerequisites for N-N coupling with or without the assistance of external reductants, (2) the structural prerequisites for N-N coupling within diiron-dinitrosyl and -mononitrosyl complexes containing the same ligand framework, and (3) the structural prerequisites with respect to the ON-NO bond distance and dihedral angle to carry out N-N coupling. Our initially reported model system utilized a dinucleating ligand with a N3O2 coordination sphere about each iron, where two NO ligands bind in a coplanar geometry. In this complex, the FeIIIFeII/FeIIFeII redox couple sits more positive than native FNORs, and a stable [hs-{Fe(NO)}7]2 complex was therefore isolated. This complex is the first reported system that undergoes unimolecular N-N coupling in the presence of one reductive equivalent, affording rapid and quantitative N2O formation. With more electron donating carboxylate ligand derivatives that enforce a N2O3 primary coordination sphere, a diferric FeIIIFeIII core can be stabilized. Here, the [hs-{Fe(NO)}7]2 complex is stable in the solid state, whereas substoichiometric N-N coupling occurs in the absence of external reductants in solution. Upon the addition of different monodentate N- and O- donor ligands to the above system, the (O)N-Fe-Fe-N(O) dihedral angle and ON-NO distance were perturbed. X-Ray crystallographic data combined with DFT modelling and N2O yields revealed that dihedral angles above 56.6o and N-N distances above 3.13 Å result in significant inhibition of N2O yields after 5 minutes. Interestingly, these complexes instead make FeII{Fe(NO)2}9/10 dinitrosyl iron complexes (DNICs) that slowly decompose over 2 hrs. Lastly, the reactivity of mono- and di-nitrosyl complexes containing the same ligand framework were compared. Here, the [hs-{Fe(NO)}7]2 complex is the second reported complex that undergoes unimolecular N-N coupling with one equivalent of reductant. The mixed-valent product of the reduction was structurally characterized, providing the first structural data on the product of N-N coupling in diiron complexes. In contrast, the corresponding hs-FeII{Fe(NO)}7 complex requires two reductive equivalents to facilitate bimolecular N-N coupling. The data discussed in this thesis outline several necessary conditions to achieve N-N coupling: (1) It can occur between two hs-{FeNO}7, (2) It more readily occurs from unimolecular coupling in a cis hs-[{FeNO}7]2 dimer, (3) Reduction of one hs-{FeNO}7 unit in the dimer leads to significant activation for N-N coupling, (4) More negative redox potentials activate hs-{FeNO}7 species, and (5) Precise geometric tuning of (O)N-N(O) distances and N-Fe-Fe-N angles are required to facilitate the reaction.