Andrew P. Hunt

Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological and pathological events in biology. Metal coordination chemistry, especially with iron, is at the heart of many biological transformations involving NO. A series of heme proteins, nitric oxide synthases (NOS), soluble guanylate cyclase (sGC), and nitrophorins, are responsible for the biosynthesis, sensing, and transport of NO. Alternatively, NO can be generated from nitrite by heme- and copper-containing nitrite reductases (NIRs). The NO-bearing small molecules such as nitrosothiols and dinitrosyl iron complexes (DNICs) can serve as an alternative vehicle for NO storage and transport. Once NO is formed, the rich reaction chemistry of NO leads to a wide variety of biological activities including reduction of NO by heme or non-heme iron-containing NO reductases and protein post-translational modifications by DNICs. Much of our understanding of the reactivity of metal sites in biology with NO and the mechanisms of these transformations has come from the elucidation of the geometric and electronic structures and chemical reactivity of synthetic model systems, in synergy with biochemical and biophysical studies on the relevant proteins themselves. This review focuses on recent advancements from studies on proteins and model complexes that not only have improved our understanding of the biological roles of NO but also have provided foundations for biomedical research and for bio-inspired catalyst design in energy science.

Cytochrome (Cyt) P450s are an important class of enzymes with numerous functions in nature. The unique reactivity of these enzymes relates to their heme b active sites with an axially bound, deprotonated cysteine (a “cysteinate”) ligand (chemically speaking a thiolate). The heme-thiolate active sites further contain a number of conserved hydrogen-bonds (H-bonds) to the bound cysteinate ligand, which have been proposed to tune and stabilize the Fe–S bond. In this work, we present the low-temperature preparation of five ferric heme-thiolate nitric oxide (NO) model complexes that contain one tunable hydrogen-bond to the bound thiolate ligand. We show that the presence of a H-bond has a dramatic effect in stabilizing the thiolate ligand against direct reaction with NO. This observation reinforces the important protective role of H-bonds in Cyt P450s. We further demonstrate that H-bond strength tunes thiolate donor strength, which, in turn, controls the N–O and Fe–NO stretching frequencies and hence, bond strengths. We observe a direct correlation between the Fe–NO and N–O stretching frequencies, indicative of a thiolate σ-trans effect (interaction). Here, very small changes in H-bond strength lead to a surprisingly large effect on the FeNO unit. This result implies that subtle changes in the Cys-pocket of a Cyt P450 can strongly affect reactivity. Importantly, using the Fe–NO/N–O correlation established here, the thiolate donor strength in heme-thiolate enzyme active sites and model complexes can be quantified in a straightforward way, using NO as a probe. This spectroscopic correlation provides a quantitative measure of the thiolate’s “push” effect, which is important in O₂-activation (Compound I formation) in Cyt P450s in general.

Despite utilizing a common cofactor binding motif, hemoproteins bearing a cysteine-derived thiolate ligand (heme-thiolate proteins) are involved in a diverse array of biological processes ranging from drug metabolism to transcriptional regulation. Though the origin of heme-thiolate functional divergence is not well understood, growing evidence suggests that the hydrogen bonding (H-bonding) environment surrounding the Fe-coordinating thiolate influences protein function. Outside of X-ray crystallography, few methods exist to characterize these critical H-bonding interactions. Electron paramagnetic resonance (EPR) spectra of heme-thiolate proteins bearing a six-coordinate, Fe(III) heme exhibit uniquely narrow low-spin (S = 1/2), rhombic signals, which are sensitive to changes in the heme-thiolate H-bonding environment. To establish a well-defined relationship between the magnitude of g-value dispersion in this unique EPR signal and the strength of the heme-thiolate H-bonding environment, we synthesized and characterized of a series of six-coordinate, aryl-thiolate-ligated Fe(III) porphyrin complexes bearing a tunable intramolecular H-bond. Spectroscopic investigation of these complexes revealed a direct correlation between H-bond strength and g-value dispersion in the rhombic EPR signal. Using density functional theory (DFT), we elucidated the electronic origins of the narrow, rhombic EPR signal in heme-thiolates, which arises from an Fe–S p_π–d_π bonding interaction. Computational analysis of the intramolecularly H-bonded heme-thiolate models revealed that H-bond donation to the coordinating thiolate reduces thiolate donor strength and weakens this Fe–S interaction, giving rise to larger g-value dispersion. By defining the relationship between heme-thiolate electronic structure and rhombic EPR signal, it is possible to compare thiolate donor strengths among heme-thiolate proteins through analysis of low-spin, Fe(III) EPR spectra. Thus, this study establishes EPR spectroscopy as a valuable tool for exploring how second coordination sphere effects influence heme-thiolate protein function.

Intravascular (IV) catheters are essential devices in the hospital that are used to monitor a patient’s blood and for administering drugs or nutrients. However, IV catheters are also prone to blood clotting at the point of insertion and infection by formation of robust bacterial biofilms on their surface. Nitric oxide (NO) is ideally suited to counteract both of these problems, due to its ability to inhibit platelet activation/aggregation and its antimicrobial properties. One way to equip catheters with NO releasing properties is by electrocatalytic nitrite reduction to NO by copper complexes in a multilumen configuration. In this work, we systematically investigate six closely related Cu(II) BMPA- and BEPA-carboxylate complexes (BMPA = bis(2-methylpyridyl)amine); BEPA = bis(2-ethylpyridyl)amine), using carboxylate groups of different chain lengths. The corresponding Cu(II) complexes were characterized using UV–vis, EPR spectroscopy, and X-ray crystallography. Using detailed cyclic voltammetry (CV) and bulk electrocatalyic studies (with real-time NO quantification), in aqueous media, pH 7.4, we are able to derive clear reactivity relations between the ligand structures of the complexes, their Faradaic efficiencies for NO generation, their turnover frequencies (TOFs), and their redox potentials. Our results show that the complex [Cu(BEPA-Bu)](OAc) is the best catalyst with a high Faradaic efficiency over large nitrite concentration ranges and the expected best tolerance to oxygen levels. For this species, the more positive reduction potential suppresses NO disproportionation, which is a major Achilles heel of the (faster) catalysts with the more negative reduction potentials.

Cyt P450 nitric oxide (NO) reductase (P450nor) is an important enzyme in fungal denitrification, responsible for the large-scale production of the greenhouse gas N₂O. In the first step of catalysis, the ferric heme-thiolate active site of P450nor binds NO to produce a ferric heme-nitrosyl or {FeNO}⁶ intermediate (in the Enemark–Feltham notation). In this paper, we present the low-temperature preparation of six new heme-thiolate {FeNO}⁶ model complexes, [Fe(TPP)(SPh*)(NO)], using a unique series of electron-poor thiophenolates (SPh*^–), and their detailed spectroscopic characterization. Our data show experimentally, for the first time, that a direct correlation exists between the thiolate donor strength and the Fe–NO and N–O bond strengths, evident from the corresponding stretching frequencies. This is due to a σ-trans effect of the thiolate ligand, which manifests itself in the population of an Fe–N–O σ-antibonding (σ*) orbital. Via control of the thiolate donor strength (using hydrogen bonds), nature is therefore able to exactly control the degree of activation of the FeNO unit in P450nor. Vice versa, NO can be used as a sensitive probe to quantify the donor strength of a thiolate ligand in a model system or protein, by simply measuring the Fe–NO and N–O frequencies of the ferric NO adduct and then projecting those data onto the correlation plot established here. Finally, we are able to show that the σ-trans effect of the thiolate is the electronic origin of the “push” effect, which is proposed to mediate O–O bond cleavage and Compound I formation in Cyt P450 monooxygenase catalysis.

Further studies aimed at examining the activity of different Cu(II)-ligand complexes to serve as electron transfer mediators to prepare novel antimicrobial/thromboresistant nitric oxide (NO) releasing intravenous catheters are reported. In these devices, the NO release can be modulated by applying different potentials or currents to reduce the Cu(II)-complexes to Cu(I) species which then reduce nitrite ions into NO(g) within a lumen of the catheter. Four different ligands are compared with respect to NO generation efficiency and stability over time using both single- and dual-lumen silicone rubber catheters: N-propanoate-N,N-bis(2-pyridylethyl)amine (BEPA-Pr), N-propanoate-N,N-bis(2-pyridylmethyl)amine (BMPA-Pr), 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3TACN), tris(2-pyridylmethyl)amine (TPMA). Of these, the Cu(II)BEPA-Pr and Cu(II)Me3TACN complexes provide biomedically useful NO fluxes from the surface of the catheters, > 2∙10-10 mol∙min-1∙cm-2, under conditions mimicking the bloodstream environment. The Cu(II)Me3TACN exhibits the best stability over time with a steady and continuous NO release observed for 8 d under a nitrogen atmosphere. Antimicrobial experiments conducted over 5 d with NO-releasing catheters turned "on" electrochemically for only 3 h or 6 h each day revealed > 2 logarithmic units in reduction of bacterial biofilm attached to the catheter surfaces. The use of optimal Cu(II)-ligand complexes within a lumen reservoir along with high levels of nitrite ions can potentially provide an effective method of preventing/decreasing the rate of infections caused by intravascular catheters.

A new portable gas phase nitric oxide (NO) generator is described for potential applications in inhaled NO (INO) therapy and during cardiopulmonary bypass (CPB) surgery. In this system, NO is produced at the surface of a large-area mesh working electrode by electrochemical reduction of nitrite ions in the presence of a soluble copper(II)-ligand electron transfer mediator complex. The NO generated is then transported into gas phase by either direct purging with nitrogen/air or via circulating the electrolyte/nitrite solution through a gas extraction silicone fiber-based membrane-dialyzer assembly. Gas phase NO concentrations can be tuned in the range of 5–1000 ppm (parts per million by volume for gaseous species), in proportion to a constant cathodic current applied between the working and counter electrodes. This new NO generation process has the advantages of rapid production times (5 min to steady-state), high Faraday NO production efficiency (ca. 93%), excellent stability, and very low cost when using air as the carrier gas for NO (in the membrane dialyzer configuration), enabling the development of potentially portable INO devices. In this initial work, the new system is examined for the effectiveness of gaseous NO to reduce the systemic inflammatory response (SIR) during CPB, where 500 ppm of NO added to the sweep gas of the oxygenator or to the cardiotomy suction air in a CPB system is shown to prevent activation of white blood cells (granulocytes and monocytes) during extracorporeal circulation with cardiotomy suction conducted with five pigs.