Amy L. Speelman

Heme versus Non-Heme Iron-Nitroxyl {FeN(H)O}8 Complexes: Electronic Structure and Biologically Relevant Reactivity

Researchers have completed extensive studies on heme and non-heme iron-nitrosyl complexes, which are labeled {FeNO}(7) in the Enemark-Feltham notation, but they have had very limited success in producing corresponding, one-electron reduced, {FeNO}(8) complexes where a nitroxyl anion (NO(-)) is formally bound to an iron(II) center. These complexes, and their protonated iron(II)-NHO analogues, are proposed key intermediates in nitrite (NO2(-)) and nitric oxide (NO) reducing enzymes in bacteria and fungi. In addition, HNO is known to have a variety of physiological effects, most notably in the cardiovascular system. HNO may also serve as a signaling molecule in mammals. For these functions, iron-containing proteins may mediate the production of HNO and serve as receptors for HNO in vivo. In this Account, we highlight recent key advances in the preparation, spectroscopic characterization, and reactivity of ferrous heme and non-heme nitroxyl (NO(-)/HNO) complexes that have greatly enhanced our understanding of the potential biological roles of these species. Low-spin (ls) heme {FeNO}(7) complexes (S = 1/2) can be reversibly reduced to the corresponding {FeNO}(8) species, which are stable, diamagnetic compounds. Because the reduction is ligand (NO) centered in these cases, it occurs at extremely negative redox potentials that are at the edge of the biologically feasible range. Interestingly, the electronic structures of ls-{FeNO}(7) and ls-{FeNO}(8) species are strongly correlated with very similar frontier molecular orbitals (FMOs) and thermodynamically strong Fe-NO bonds. In contrast, high-spin (hs) non-heme {FeNO}(7) complexes (S = 3/2) can be reduced at relatively mild redox potentials. Here, the reduction is metal-centered and leads to a paramagnetic (S = 1) {FeNO}(8) complex. The increased electron density at the iron center in these species significantly decreases the covalency of the Fe-NO bond, making the reduced complexes highly reactive. In the absence of steric bulk, monomeric high-spin {FeNO}(8) complexes decompose rapidly. Notably, in a recently prepared, dimeric [{FeNO}(7)]2 species, we observed that reduction leads to rapid N-N bond formation and N2O generation, which directly models the reactivity of flavodiiron NO reductases (FNORs). We have also made key progress in the preparation and stabilization of corresponding HNO complexes, {FeNHO}(8), using both heme and non-heme ligand sets. In both cases, we have taken advantage of sterically bulky coligands to stabilize these species. ls-{FeNO}(8) complexes are basic and easily form corresponding ls-{FeNHO}(8) species, which, however, decompose rapidly via disproportionation and H2 release. Importantly, we recently showed that we can suppress this reaction via steric protection of the bound HNO ligand. As a result, we have demonstrated that ls-{FeNHO}(8) model complexes are stable and amenable to spectroscopic characterization. Neither ls-{FeNO}(8) nor ls-{FeNHO}(8) model complexes are active for N-N coupling, and hence, seem unsuitable as reactive intermediates in nitric oxide reductases (NORs). Hs-{FeNO}(8) complexes are more basic than their hs-{FeNO}(7) precursors, but their electronic structure and reactivity is not as well characterized.

Characterization of a High‐Spin Non‐Heme {FeNO}8 Complex: Implications for the Reactivity of Iron Nitroxyl Species in Biology

Stable but able: Chemical and electrochemical reduction of a five-coordinate high-spin non-heme {FeNO}(7) complex (see structure: N blue, Fe orange, and O red) generated the first stable high-spin (S=1) non-heme {FeNO}(8) model complex. The finding that the reduction is metal-centered and causes a decrease in FeNO covalency indicates that in biological systems, reduction activates stable non-heme FeNO units for further transformations.

Unusual Synthetic Pathway for an {Fe(NO)2}9 Dinitrosyl Iron Complex (DNIC) and Insight into DNIC Electronic Structure via Nuclear Resonance Vibrational Spectroscopy

Dinitrosyl iron complexes (DNICs) are among the most abundant NO-derived cellular species. Monomeric DNICs can exist in the {Fe(NO)2}(9) or {Fe(NO)2}(10) oxidation state (in the Enemark-Feltham notation). However, experimental studies of analogous DNICs in both oxidation states are rare, which prevents a thorough understanding of the differences in the electronic structures of these species. Here, the {Fe(NO)2}(9) DNIC [Fe(dmp)(NO)2](OTf) (1; dmp = 2,9-dimethyl-1,10-phenanthroline) is synthesized from a ferrous precursor via an unusual pathway, involving disproportionation of an {FeNO}(7) complex to yield the {Fe(NO)2}(9) DNIC and a ferric species, which is subsequently reduced by NO gas to generate a ferrous complex that re-enters the reaction cycle. In contrast to most {Fe(NO)2}(9) DNICs with neutral N-donor ligands, 1 exhibits high solution stability and can be characterized structurally and spectroscopically. Reduction of 1 yields the corresponding {Fe(NO)2}(10) DNIC [Fe(dmp)(NO)2] (2). The Mössbauer isomer shift of 2 is 0.08 mm/s smaller than that of 1, which indicates that the iron center is slightly more oxidized in the reduced complex. The nuclear resonance vibrational spectra (NRVS) of 1 and 2 are distinct and provide direct experimental insight into differences in bonding in these complexes. In particular, the symmetric out-of-plane Fe-N-O bending mode is shifted to higher energy by 188 cm(-1) in 2 in comparison to 1. Using quantum chemistry centered normal coordinate analysis (QCC-NCA), this is shown to arise from an increase in Fe-NO bond order and a stiffening of the Fe(NO)2 unit upon reduction of 1 to 2. DFT calculations demonstrate that the changes in bonding arise from an iron-centered reduction which leads to a distinct increase in Fe-NO π-back-bonding in {Fe(NO)2}(10) DNICs in comparison to the corresponding {Fe(NO)2}(9) complexes, in agreement with all experimental findings. Finally, the implications of the electronic structure of DNICs for their reactivity are discussed, especially with respect to N-N bond formation in NO reductases.

Structural and Spectroscopic Characterization of a High‐Spin {FeNO}6 Complex with an Iron(IV)−NO− Electronic Structure

Although the interaction of low-spin ferric complexes with nitric oxide has been well studied, examples of stable high-spin ferric nitrosyls (such as those that could be expected to form at typical non-heme iron sites in biology) are extremely rare. Using the TMG3 tren co-ligand, we have prepared a high-spin ferric NO adduct ({FeNO}(6) complex) via electrochemical or chemical oxidation of the corresponding high-spin ferrous NO {FeNO}(7) complex. The {FeNO}(6) compound is characterized by UV/Visible and IR spectroelectrochemistry, Mössbauer and NMR spectroscopy, X-ray crystallography, and DFT calculations. The data show that its electronic structure is best described as a high-spin iron(IV) center bound to a triplet NO(-) ligand with a very covalent iron-NO bond. This finding demonstrates that this high-spin iron nitrosyl compound undergoes iron-centered redox chemistry, leading to fundamentally different properties than corresponding low-spin compounds, which undergo NO-centered redox transformations.

The Semireduced Mechanism for Nitric Oxide Reduction by Non-Heme Diiron Complexes: Modeling Flavodiiron Nitric Oxide Reductases

Flavodiiron nitric oxide reductases (FNORs) are a subclass of flavodiiron proteins (FDPs) capable of preferential binding and subsequent reduction of NO to N2O. FNORs are found in certain pathogenic bacteria, equipping them with resistance to nitrosative stress, generated as a part of the immune defense in humans, and allowing them to proliferate. Here, we report the spectroscopic characterization and detailed reactivity studies of the diiron dinitrosyl model complex [Fe2(BPMP)(OPr)(NO)2](OTf)2 for the FNOR active site that is capable of reducing NO to N2O [Zheng et al., J. Am. Chem. Soc. 2013, 135, 4902-4905]. Using UV-vis spectroscopy, cyclic voltammetry, and spectro-electrochemistry, we show that one reductive equivalent is in fact sufficient for the quantitative generation of N2O, following a semireduced reaction mechanism. This reaction is very efficient and produces N2O with a first-order rate constant k > 102 s-1. Further isotope labeling studies confirm an intramolecular N-N coupling mechanism, consistent with the rapid time scale of the reduction and a very low barrier for N-N bond formation. Accordingly, the reaction proceeds at -80 °C, allowing for the direct observation of the mixed-valent product of the reaction. At higher temperatures, the initial reaction product is unstable and decays, ultimately generating the diferrous complex [Fe2(BPMP)(OPr)2](OTf) and an unidentified ferric product. These results combined offer deep insight into the mechanism of NO reduction by the relevant model complex [Fe2(BPMP)(OPr)(NO)2]2+ and provide direct evidence that the semireduced mechanism would constitute a highly efficient pathway to accomplish NO reduction to N2O in FNORs and in synthetic catalysts.

Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet-visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

CCDC 1841320: Experimental Crystal Structure Determination

Related Article: Amy L. Speelman, Corey J. White, Bo Zhang, E. Ercan Alp, Jiyong Zhao, Michael Hu, Carsten Krebs, James Penner-Hahn, Nicolai Lehnert|2018|J.Am.Chem.Soc.|140||doi:10.1021/jacs.8b06095