Eunsuk Kim

Superoxo, μ-peroxo, and μ-oxo complexes from heme/O2 and heme-Cu/O2 reactivity: Copper ligand influences in cytochrome c oxidase models

The O2-reaction chemistry of 1:1 mixtures of (F8)FeII (1; F8 = tetrakis(2,6-diflurorophenyl)porphyrinate) and [(LMe2N)CuI]+ (2; LMe2N = N,N-bis{2-[2-(N′,N′-4-dimethylamino)pyridyl]ethyl}methylamine) is described, to model aspects of the chemistry occurring in cytochrome c oxidase. Spectroscopic investigations, along with stopped-flow kinetics, reveal that low-temperature oxygenation of 1/2 leads to rapid formation of a heme-superoxo species (F8)FeIII-(O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document}) (3), whether or not 2 is present. Complex 3 subsequently reacts with 2 to form [(F8)FeIII–(O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{2-}}}\end{equation*}\end{document})–CuII(LMe2N)]+ (4), which thermally converts to [(F8)FeIII–(O)–CuII(LMe2N)]+ (5), which has an unusually bent (Fe–O–Cu) bond moiety. Tridentate chelation, compared with tetradentate, is shown to dramatically lower the ν(O–O) values observed in 4 and give rise to the novel structural features in 5.

Phenol Nitration Induced by an {Fe(NO)2}10 Dinitrosyl Iron Complex

Cellular dinitrosyl iron complexes (DNICs) have long been considered NO carriers. Although other physiological roles of DNICs have been postulated, their chemical functionality outside of NO transfer has not been demonstrated thus far. Here we report the unprecedented dioxygen reactivity of a N-bound {Fe(NO)(2)}(10) DNIC, [Fe(TMEDA)(NO)(2)] (1). In the presence of O(2), 1 becomes a nitrating agent that converts 2,4,-di-tert-butylphenol to 2,4-di-tert-butyl-6-nitrophenol via formation of a putative iron-peroxynitrite [Fe(TMEDA)(NO)(ONOO)] (2) that is stable below -80 °C. Iron K-edge X-ray absorption spectroscopy on 2 supports a five-coordinated metal center with a bound peroxynitrite in a cyclic bidentate fashion. The peroxynitrite ligand of 2 readily decays at increased temperature or under illumination. These results suggest that DNICs could have multiple physiological or deleterious roles, including that of cellular nitrating agents.

A bifunctional platinum(II) antitumor agent that forms DNA adducts with affinity for the estrogen receptor

A strategy is described for the re-design of DNA damaging platinum(II) complexes to afford elevated toxicity towards cancer cells expressing the estrogen receptor (ER). Two platinum-based toxicants are described in which a DNA damaging warhead, [Pt(en)Cl(2)] (en, ethylenediamine), is tethered to either of two functional groups. The first agent, [6-(2-amino-ethylamino)-hexyl]-carbamic acid 2-[6-(7alpha-estra-1,3,5,(10)-triene)-hexylamino]-ethyl ester platinum(II) dichloride ((Est-en)PtCl(2)), terminates in a ligand for the ER. The second agent is a control compound lacking the steroid; this compound, N-[6-(2-amino-ethylamino)-hexyl]-benzamide platinum(II) dichloride ((Bz-en)PtCl(2))), terminates in a benzamide moiety, which lacks affinity for the ER. Using a competitive binding assay, Est-en had 28% relative binding affinity (RBA) for the ER as compared to 17beta-estradiol. After covalent binding to a synthetic DNA duplex 16-mer, the compound retained its affinity for the ER; specificity of the binding event was demonstrated by the ability of free 17beta-estradiol as a competitor to disrupt the DNA adduct-ER complex. The (Est-en)PtCl(2) compound showed higher toxicity against the ER positive ovarian cancer cell line CAOV3 than did the control compound. (Est-en)PtCl(2) was also more toxic to the ER positive breast cancer line, MCF-7, than to an ER negative line, MDA-MB231.

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.

Nitric oxide reactivity of [2Fe-2S] clusters leading to H2S generation.

The crosstalk between two biologically important signaling molecules, nitric oxide (NO) and hydrogen sulfide (H2S), proceeds via elusive mechanism(s). Herein we report the formation of H2S by the action of NO on synthetic [2Fe-2S] clusters when the reaction environment is capable of providing a formal H(•) (e(-)/H(+)). Nitrosylation of (NEt4)2[Fe2S2(SPh)4] (1) in the presence of PhSH or (t)Bu3PhOH results in the formation of (NEt4)[Fe(NO)2(SPh)2] (2) and H2S with the concomitant generation of PhSSPh or (t)Bu3PhO(•). The amount of H2S generated is dependent on the electronic environment of the [2Fe-2S] cluster as well as the type of H(•) donor. Employment of clusters with electron-donating groups or H(•) donors from thiols leads to a larger amount of H2S evolution. The 1/NO reaction in the presence of PhSH exhibits biphasic decay kinetics with no deuterium kinetic isotope effect upon PhSD substitution. However, the rates of decay increase significantly with the use of 4-MeO-PhSH or 4-Me-PhSH in place of PhSH. These results provide the first chemical evidence to suggest that [Fe-S] clusters are likely to be a site for the crosstalk between NO and H2S in biology.

Synthetic modeling chemistry of iron-sulfur clusters in nitric oxide signaling.

Nitric oxide (NO) is an important signaling molecule that is involved in many physiological and pathological functions. Iron-sulfur proteins are one of the main reaction targets for NO, and the [Fe-S] clusters within these proteins are converted to various iron nitrosyl species upon reaction with NO, of which dinitrosyl iron complexes (DNICs) are the most prevalent. Much progress has been made in identifying the origin of cellular DNIC generation. However, it is not well-understood which other products besides DNICs may form during [Fe-S] cluster degradation nor what effects DNICs and other degradation products can have once they are generated in cells. Even more elusive is an understanding of the manner by which cells cope with unwanted [Fe-S] modifications by NO. This Account describes our synthetic modeling efforts to identify cluster degradation products derived from the [2Fe-2S]/NO reaction in order to establish their chemical reactivity and repair chemistry. Our intent is to use the chemical knowledge that we generate to provide insight into the unknown biological consequences of cluster modification. Our recent advances in three different areas are described. First, new reaction conditions that lead to the formation of previously unrecognized products during the reaction of [Fe-S] clusters with NO are identified. Hydrogen sulfide (H2S), a gaseous signaling molecule, can be generated from the reaction between [2Fe-2S] clusters and NO in the presence of acid or formal H• (e(-)/H(+)) donors. In the presence of acid, a mononitrosyl iron complex (MNIC) can be produced as the major iron-containing product. Second, cysteine analogues can efficiently convert MNICs back to [2Fe-2S] clusters without the need for any other reagents. This reaction is possible for cysteine analogues because of their ability to labilize NO from MNICs and their capacity to undergo C-S bond cleavage, providing the necessary sulfide for [2Fe-2S] cluster formation. Lastly, unique dioxygen reactivity of various types of DNICs has been established. N-bound neutral {Fe(NO)2}(10) DNICs react with O2 to generate low-temperature stable peroxynitrite (ONOO(-)) species, which then carry out nitration chemistry in the presence of phenolic substrates, relevant to tyrosine nitration chemistry. The reaction between S-bound anionic {Fe(NO)2}(9) DNICs and O2 results in the formation of Roussins red esters (RREs) and thiol oxidation products, chemistry that may be important in biological cysteine oxidation. The N-bound cationic {Fe(NO)2}(9) DNICs can spontaneously release NO, and this property can be utilized in developing a new class of NO-donating agents with anti-inflammatory activity.

Transformation of a mononitrosyl iron complex to a [2Fe-2S] cluster by a cysteine analogue.

Reversible modification of iron-sulfur clusters by nitric oxide acts as a genetic switch in a group of regulatory proteins. While the conversion of [Fe-S] clusters to iron-nitrosyls has been widely studied in the past, little is known about the reverse process, the repair of [Fe-S] clusters. Reported here is a system in which a mononitrosyl iron complex (MNIC), (PPN)[Fe(S(t)Bu)3(NO)] (1), is converted to a [2Fe-2S] cluster, (PPN)2[Fe2S2(SCH2CH2C(O)OMe)4] (2). This conversion requires only the addition of a cysteine analogue, 3-mercaptomethylpropionate (MMP), at room temperature without the need for any other reagents. The identity of 2 was confirmed spectroscopically, chemically, crystallographically, and analytically. Mass spectrometry and (34)S labeling studies support that the bridging sulfides in 2 derive from the added MMP, the cysteine analogue. The NO lost during the conversion of 1 to 2 is trapped in a dinitrosyl iron side product, (PPN)[Fe(SCH2CH2C(O)OMe)2(NO)2] (4). The present system implies that MNICs are likely intermediates in the repair of NO-damaged [2Fe-2S] clusters and that cysteine is a viable molecule responsible for the destabilization of MINCs and the formation of [2Fe-2S] clusters.

Coordination-triggered NO release from a dinitrosyl iron complex leads to anti-inflammatory activity

Dinitrosyl iron complexes (DNICs) are widely considered NO storage and donor molecules in cells. However, what induces an NO release from iron in DNICs and the subsequent biological consequences remain elusive. The chemistry and biology of the NO release activity of DNICs are reported here. Changes in redox status or coordination number of discrete N-bound DNICs, respectively [Fe(TMEDA)(NO)2] (1) and [Fe(TMEDA)(NO)2I] (2), can generate a metastable {Fe(NO)2}9 DNIC, [Fe(TMEDA)(NO)2]+, with νNO at 1769 and 1835 cm−1 and an EPR signal at g = 2.04, that spontaneously releases NO in solution. The NO release activity of 2 results in the up- and downregulation of heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS), respectively, in murine RAW 264.7 macrophages. Furthermore, treatment with 2 leads to downregulation of pro-inflammatory cytokines, TNF-α and IL-6, and upregulation of the anti-inflammatory cytokine, IL-10. Taken together, these results demonstrate that the appropriate control of redox and coordination chemistry of DNICs could enable them to become anti-inflammatory agents, suggesting a potential new role for cellular DNICs.

A DNA-Based Nanomechanical Device Used To Characterize the Distortion of DNA by Apo-SoxR Protein

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple-crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In our case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to ~2 bp. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding of the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

New Synthetic Routes to Iron–Sulfur Clusters: Deciphering the Repair Chemistry of [2Fe–2S] Clusters from Mononitrosyl Iron Complexes

The nitrosylation of inorganic protein cofactors, specifically that of [Fe-S] clusters to form iron nitrosyls, plays a number of important roles in biological systems. In some of these cases, it is expected that a repair process reverts the nitrosylated iron species to intact [Fe-S] clusters. The repair of nitrosylated [2Fe-2S] cluster, primarily in the form of protein-bound dinitrosyl iron complexes (DNICs), has been observed in vitro and in vivo, but the mechanism of this process remains uncertain. The present work expands upon a previous observation (Fitzpatrick et al. J. Am. Chem. Soc. 2014, 136, 7229) of the ability of mononitrosyl iron complexes (MNICs) to be converted into [2Fe-2S] clusters by the addition of nothing other than a cysteine analogue. Herein, each of the critical elementary steps in the cluster repair has been dissected to elucidate the roles of the cysteine analogue. Systematic variations of a cysteine analogue employed in the repair reaction suggest that (i) the bidentate coordination of a cysteine analogue to MNIC promotes NO release from iron, and (ii) deprotonation of the α carbon of the ferric-bound cysteine analogue leads to the C-S cleavage en route to the formation of [2Fe-2S] cluster. The [2Fe-2S] cluster bearing a cysteine analogue has also been synthesized from thiolate-bridged iron dimers of the form [Fe2(μ-SR)2(SR)4](0/2-), which implies that such species may be present as intermediates in the cluster repair. In addition to MNICs, mononuclear tetrathiolate ferric or ferrous species have been established as another form of iron from which [2Fe-2S] clusters can be generated without need for any other reagent but a cysteine analogue. The results of these experiments bring to light new chemistry of classic coordination complexes and provides further insight into the repair of NO-modified [2Fe-2S] clusters.