Spectroscopic, electrochemical, and kinetic trends in Fe(III)-thiolate disproportionation near physiologic pH.

In addition to its primary oxygen-atom-transfer function, cysteamine dioxygenase (ADO) exhibits a relatively understudied anaerobic disproportionation reaction (ADO-Fe(III)-SR → ADO-Fe(II) + ½ RSSR) with its native substrates. Inspired by ADO disproportionation reactivity, we employ [Fe(tacn)Cl3] (tacn = 1,4,7-triazacyclononane) as a precursor for generating Fe(III)-thiolate model complexes in buffered aqueous media. A series of Fe(III)-thiolate model complexes are generated in situ using aqueous [Fe(tacn)Cl3] and thiol-containing ligands cysteamine, penicillamine, mercaptopropionate, cysteine, cysteine methyl ester, N-acetylcysteine, and N-acetylcysteine methyl ester. We observe trends in UV-Vis and electron paramagnetic resonance (EPR) spectra, disproportionation rate constants, and cathodic peak potentials as a function of thiol ligand. These trends will be useful in rationalizing substrate-dependent Fe(III)-thiolate disproportionation reactions in metalloenzymes.

The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo-, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo- on DNA-modified electrodes, a large cathodic signal centered at -140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2COREexo-, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]2+, to oxidized Pol2COREexo- bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo-. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo- via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation.

Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)2Fe(NO)2]-, and Roussin's red ester, [(µ-Cys)2Fe2(NO)4], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin's red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with 14N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin's red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with 14NO and 15NO support the assignment of a protein-bound [(µ-Cys)2Fe2(NO)4] Roussin's red ester. The [Fe4S4]2+/3+ redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 ± 6 and -674 ± 8 mV vs NHE, respectively, corresponding to a shift of approximately -800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.

The Role of Coordination Environment and pH in Tuning the Oxidation Rate of Europium(II).

The EuII/III redox couple offers metal-based oxidation-sensing with magnetic resonance imaging making the study of EuII oxidation chemistry important in the design of new probes. Accordingly, we explored oxidation reactions with a set of EuII -containing complexes. Superoxide formation from the reaction between EuII and dioxygen was observed using electron paramagnetic resonance spectroscopy. Additionally, oxidation kinetics of three EuII -containing complexes with bromate and glutathione disulfide at pH values, including 5 and 7, is reported. In the reaction with bromate, the oxidation rate of two of the complexes increased by 7.3 and 6.7 times upon decreasing pH from 7 to 5, but the rate increased by 17 times for a complex containing amide functional groups over the same pH range. The oxidation rate of a fluorobenzo-functionalized cryptate was relatively slow, indicating that the ligand used to impart thermodynamic oxidative stability might also be useful for controlling oxidation kinetics.

Electrochemical investigation of the Eu3+/2+ redox couple in complexes with variable numbers of glycinamide and acetate pendant arms.

The Eu3+/2+ redox couple provides a convenient design platform for responsive pO2 sensors for magnetic resonance imaging (MRI). Specifically the Eu2+ ion provides T1w contrast enhancement under hypoxic conditions in tissues, whereas, under normoxia, the Eu3+ ion can produce contrast from chemical exchange saturation transfer in MRI. The oxidative stability of the Eu3+/2+ redox couple for a series of tetraaza macrocyclic complexes was investigated in this work using cyclic voltammetry. A series of Eu-containing cyclen-based macrocyclic complexes revealed positive shifts in the Eu3+/2+ redox potentials with each replacement of a carboxylate coordinating arm of the ligand scaffold with glycinamide pendant arms. The data obtained reveal that the complex containing four glycinamide coordinating pendant arms has the highest oxidative stability of the series investigated.

Spectroscopic Characterization of the 3+ and 2+ Oxidation States of Europium in a Macrocyclic Tetraglycinate Complex.

The 3+ and 2+ oxidation states of europium have drastically different magnetic and spectroscopic properties. Electrochemical measurements are often used to probe EuIII/II oxidation state changes, but a full suite of spectroscopic characterization is necessary to demonstrate conversion between these two oxidation states in solution. Here, we report the facile conversion of an europium(III) tetraglycinate complex into its EuII analogue. We present electrochemical, luminescence, electron paramagnetic resonance, UV-visible, and NMR spectroscopic data demonstrating complete reversibility from the reduction and oxidation of the 3+ and 2+ oxidation states, respectively. The EuII-containing analogue has kinetic stability within the range of clinically approved GdIII-containing complexes using an acid-catalyzed dissociation experiment. Additionally, we demonstrate that the 3+ and 2+ oxidation states provide redox-responsive behavior through chemical-exchange saturation transfer or proton relaxation, respectively. These results will be applicable to a wide range of redox-responsive contrast agents and Eu-containing complexes.

A Eu(II)-Containing Cryptate as a Redox Sensor in Magnetic Resonance Imaging of Living Tissue.

The Eu(II) ion rivals Gd(III) in its ability to enhance contrast in magnetic resonance imaging. However, all reported Eu(II)-based complexes have been studied in vitro largely because the tendency of Eu(II) to oxidize to Eu(III) has been viewed as a major obstacle to in vivo imaging. Herein, we present solid- and solution-phase characterization of a Eu(II)-containing cryptate and the first in vivo use of Eu(II) to provide contrast enhancement. The results indicate that between one and two water molecules are coordinated to the Eu(II) core upon dissolution. We also demonstrate that Eu(II)-based contrast enhancement can be observed for hours in a mouse.

Evaluation of Eu(II) -based positive contrast enhancement after intravenous, intraperitoneal, and subcutaneous injections.

Eu(II) -based contrast agents offer physiologically relevant, metal-based redox sensing that is unachievable with Gd(III) -based contrast agents. To evaluate the in vivo contrast enhancement of Eu(II) as a function of injection type, we performed intravenous, intraperitoneal, and subcutaneous injections in mice. Our data reveal a correlation between reported oxygen content and expected rates of diffusion with the persistence of Eu(II) -based contrast enhancement. Biodistribution studies revealed europium clearance through the liver and kidneys for intravenous and intraperitoneal injections, but no contrast enhancement was observed in organs associated with clearance. These data represent a step toward understanding the behavior of Eu(II) -based complexes in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Overcoming the concentration-dependence of responsive probes for magnetic resonance imaging.

In magnetic resonance imaging, contrast agents are molecules that increase the contrast-to-noise ratio of non-invasively acquired images. The information gained from magnetic resonance imaging can be increased using responsive contrast agents that undergo chemical changes, and consequently changes to contrast enhancement, for example in response to specific biomarkers that are indicative of diseases. A major limitation with modern responsive contrast agents is concentration-dependence that requires the concentration of contrast agent to be known: an extremely challenging task in vivo. Here, we review advances in several strategies aimed at overcoming the concentration-dependent nature of responsive contrast agents.

Oxidation-responsive Eu(2+/3+)-liposomal contrast agent for dual-mode magnetic resonance imaging.

An oxidation-responsive contrast agent for magnetic resonance imaging was synthesized using Eu(2+) and liposomes. Positive contrast enhancement was observed with Eu(2+), and chemical exchange saturation transfer was observed before and after oxidation of Eu(2+). Orthogonal detection modes render the concentration of Eu inconsequential to molecular information provided through imaging.