Reducing O2 sensitivity in electrochemical nitric oxide releasing catheters: An O2-tolerant copper(II)-ligand nitrite reduction catalyst and a glucose oxidase catheter coating.

Electrocatalytic nitric oxide (NO) generation from nitrite (NO2-) within a single lumen of a dual-lumen catheter using CuII-ligand (CuII-L) mediators have been successful at demonstrating NO's potent antimicrobial and antithrombotic properties to reduce bacterial counts and mitigate clotting under low oxygen conditions (e.g., venous blood). Under more aerobic conditions, the O2 sensitivity of the Cu(II)-ligand catalysts and the reaction of O2 (highly soluble in the catheter material) with the NO diffusing through the outer walls of the catheters results in a large decreases in NO fluxes from the surfaces of the catheters, reducing the utility of this approach. Herein, we describe a new more O2-tolerant CuII-L catalyst, [Cu(BEPA-EtSO3)(OTf)], as well as a potentially useful immobilized glucose oxidase enzyme-coating approach that greatly reduces the NO reactivity with oxygen as the NO partitions and diffuses through the catheter material. Results from this work demonstrate that very effective NO fluxes (>1*10-10 mol min-1 cm-2) from a single-lumen silicone rubber catheter can be achieved in the presence of up to 10% O2 saturated solutions.

Electrochemical Generation of Nitric Oxide for Medical Applications.

Over the past 30 years, the significance of nitric oxide (NO) has become increasingly apparent in mammalian physiology. It is biosynthesized by three isoforms of nitric oxide synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Neuronal and eNOS both produce low levels of NO (nM) as a signaling agent and vasodilator, respectively. Inducible (iNOS) is present in activated macrophages at sites of infection to generate acutely toxic (µM) levels of NO as part of the mammalian immune defense mechanism. These discoveries have led to numerous animal and clinical studies to evaluate the potential therapeutic utility of NO in various medical operations/treatments, primarily using NO gas (via gas-cylinders) as the NO source. In this review, we focus specifically on recent advances in the electrochemical generation of NO (E-NOgen) as an alternative means to generate NO from cheap and inert sources, and the fabrication and testing of biomedical devices that utilize E-NOgen to controllably generate NO for medical applications.

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.

A Monohydrosulfidodinitrosyldiiron Complex That Generates N2O as a Model for Flavodiiron Nitric Oxide Reductases: Reaction Mechanism and Electronic Structure.

Flavodiiron nitric oxide reductases (FNORs) protect microbes from nitrosative stress under anaerobic conditions by mediating the reduction of nitric oxide (NO) to nitrous oxide (N2O). The proposed mechanism for the catalytic reduction of NO by FNORs involves a dinitrosyldiiron intermediate with a [hs-{FeNO}7]2 formulation, which produces N2O and a diferric species. Moreover, both NO and hydrogen sulfide (H2S) have been implicated in several similar physiological functions in biology and are also known to cross paths in cell signaling. Here we report the synthesis, spectroscopic and theoretical characterization, and N2O production activity of an unprecedented monohydrosulfidodinitrosyldiiron compound, with a [(HS)hs-{FeNO}7/hs-{FeNO}7] formulation, that models the key dinitrosyl intermediate of FNORs. The generation of N2O from this unique compound follows a semireduced pathway, where one-electron reduction generates a reactive hs-{FeNO}8 center via the occupation of an Fe-NO antibonding orbital. In contrast to the well-known reactivity of H2S and NO, the coordinated hydrosulfide remains unreactive toward NO and acts only as a spectator ligand during the NO reduction process.

Functional Models for the Mono- and Dinitrosyl Intermediates of FNORs: Semireduction versus Superreduction of NO.

The reduction of NO to N2O by flavodiiron nitric oxide reductases (FNORs) is related to the disruption of the defense mechanism in mammals against invading pathogens. The proposed mechanism for this catalytic reaction involves both nonheme mono- and dinitrosyl diiron(II) species as the key intermediates. Recently, we reported an initial account for NO reduction activity of an unprecedented mononitrosyl diiron(II) complex, [Fe2(N-Et-HPTB)(NO)(DMF)3](BF4)3 (1) (N-Et-HPTB is the anion of N,N,N',N'-tetrakis(2-(l-ethylbenzimidazolyl))-2-hydroxy-1,3-diaminopropane; DMF = dimethylformamide) with [FeII{FeNO}7] formulation [Jana et al. J. Am. Chem. Soc. 2017, 139, 14380]. Here we report the full account for the selective synthesis, characterization, and reactivity of FNOR model complexes, which include a dinitrosyl diiron(II) complex, [Fe2(N-Et-HPTB)(NO)2(DMF)2](BF4)3 (2) with [{FeNO}7]2 formulation and a related, mixed-valent diiron(II, III) complex, [Fe2(N-Et-HPTB)(OH)(DMF)3](BF4)3 (3). Importantly, whereas complex 2 is able to produce 89% of N2O via a semireduced mechanism (1 equiv of CoCp2 per dimer = 50% of NO reduced), complex 1, under the same conditions (0.5 equiv of CoCp2 per dimer = 50% of NO reduced), generates only ∼50% of N2O. The mononitrosyl complex therefore requires superreduction for quantitative N2O generation, which constitutes an interesting dichotomy between 1 and 2. Reaction products obtained after N2O generation by 2 using 1 and 2 equiv of reductant were characterized by molecular structure determination and electron paramagnetic resonance spectroscopy. Despite several available literature reports on N2O generation by diiron complexes, this is the first case where the end products from these reactions could be characterized unambiguously, which clarifies a number of tantalizing observations about the nature of these products in the literature.

Non-Heme Diiron Model Complexes Can Mediate Direct NO Reduction: Mechanistic Insight into Flavodiiron NO Reductases.

Flavodiiron nitric oxide reductases (FNORs), a common enzyme family found in various types of pathogenic bacteria, are capable of reducing nitric oxide (NO) to nitrous oxide (N2O) as a protective detoxification mechanism. Utilization of FNORs in pathogenic bacteria helps them survive and proliferate in the human body, thus causing chronic infections. In this paper, we present a new diiron model complex, [Fe2((Py2PhO2)MP)(OPr)2](OTf), with bridging propionate ligands (OPr-) that is capable of directly reducing NO to N2O in quantitative yield without the need to (super)reduce the complex. We first prepared the diferric precursor and characterized it by UV-vis, IR, NMR and Mössbauer spectroscopies, cyclic voltammetry, and mass spectrometry. This complex can then conveniently be reduced to the diferrous complex using CoCp2. Even though this diferrous complex is highly reactive, we have successfully isolated and characterized this species using X-ray crystallography and various spectroscopic techniques. Most importantly, upon reacting this diferrous complex with NO gas, we observe quantitative formation of N2O via IR gas headspace analysis, the first demonstration of direct NO reduction by a non-heme diiron model complex. This finding directly supports recent mechanistic proposals for FNORs.

Non-heme High-Spin {FeNO}6-8 Complexes: One Ligand Platform Can Do It All.

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 TMG3tren coligand. Redox transformations from the hs-{FeNO}7 complex [Fe(TMG3tren)(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 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.

A Structural Model for the Iron-Nitrosyl Adduct of Gentisate Dioxygenase.

We present the synthesis, properties, and characterization of [Fe(T1Et4iPrIP)(NO)(H2O)2](OTf)2 (1) (T1Et4iPrIP = Tris(1-ethyl-4-isopropyl-imidazolyl)phosphine) as a model for the nitrosyl adduct of gentisate 1,2-dioxygenase (GDO). The further characterization of [Fe(T1Et4iPrIP)(THF)(NO)(OTf)](OTf) (2) which was previously communicated (Inorg. Chem. 2014, 53, 5414) is also presented. The weighted average Fe-N-O angle of 162° for 1 is very close to linear (≥ 165°) for these types of complexes. The coordinated water ligands participate in hydrogen bonding interactions. The spectral properties (EPR, UV-vis, FTIR) for 1 are compared with 2 and found to be quite comparable. Complex 1 closely follows the relationship between the Fe-N-O angle and NO vibrational frequency which was previously identified for 6-coordinate {FeNO}7 complexes. Liquid FTIR studies on 2 indicate that the ν(NO) vibration position is sensitive to solvent shifting to lower energy (relative to the solid) in donor solvent THF and shifting to higher energy in dichloromethane. The basis for this behavior is discussed. The K eq for NO binding in 2 was calculated in THF and found to be 470 M-1. Density functional theory (DFT) studies on 1 indicate donation of electron density to the iron center from the π* orbitals of formally NO-. Such a donation accounts for the near linearity of the Fe-N-O bond and the large ν(NO) value of 1791 cm-1.

Functional Mononitrosyl Diiron(II) Complex Mediates the Reduction of NO to N2O with Relevance for Flavodiiron NO Reductases.

Reaction of [Fe2(N-Et-HPTB)(CH3COS)](BF4)2 (1) with (NO)(BF4) produces a nonheme mononitrosyl diiron(II) complex, [Fe2(N-Et-HPTB)(NO)(DMF)3](BF4)3 (2). Complex 2 is the first example of a [FeII{Fe(NO)}7] species and is also the first example of a mononitrosyl diiron(II) complex that mediates the reduction of NO to N2O. This work describes the selective synthesis, detailed characterization and NO reduction activity of 2 and thus provides new insights regarding the mechanism of flavodiiron nitric oxide reductases.