The mechanism of proton transfer (PT), and how it is affected by water structure, is a fundamental issue in numerous chemical and biological processes. Formulated more than 200 years ago, a possible model for PT in aqueous media was proposed by Grotthuss, which continues to be actively studied and debated. In this study, we exploit electron paramagnetic resonance to investigate PT in aqueous solutions. Our proposed method employs pH-sensitive stable nitroxyl radicals and makes use of photolysis of 2-nitrobenzaldehyde to generate protons in the sub-nanosecond timescale. This approach was used to study the impact of classical chaotropic compounds on PT as studied in various aqueous solutions, i.e. 8 M urea, 6 M guanidine hydrochloride (Gdn·HCl), and potassium chloride (KCl). Our findings confirm significant impacts on PT rates. For instance, in 6 M Gdn·HCl, PT occurred 40-fold slower than in water. The method's sensitivity to water structure is demonstrated, highlighting its potential for monitoring the kinetics of PT in ice and in proteins.

We earlier showed that Torpedo californica acetylcholinesterase (AChE) contains a cluster of four conserved aspartates that can strongly bind divalent cations, which we named the 4D motif. Binding of the divalent metal cations greatly increases its thermal stability. Here we systematically examined all available crystallographic structures of T. californica AChE. Two additional metal-binding sites were identified, both composed of acidic and histidine residues. Relative binding to the 4D and additional sites was studied using metadynamics simulations. It was observed that in crystal structures devoid of metal ions in the 4D site, the conformation of T. californica AChE is almost identical to that in structures in which it is occupied by a divalent metal ion. Closer examination of the 4D motif reveals that three of the four acidic residues form ion pairs with conserved basic residues surrounding them. We named this new motif the 4A/3B motif. Molecular dynamics with quantum potential simulations was used to quantify the 4D motif's binding strength compared with that of the metal-binding site in the protein fXIIIa, which consists of four aspartates, but is devoid of adjacent cationic residues. Whereas fXIIIa's 4D site, in the absence of a metal cation, expanded significantly in the simulation, that of Torpedo AChE displayed only minor periodic changes in size. Furthermore, the energy of metal ion unbinding from the two sites differs by ca. 10 kcal/mol. We identified several other proteins in the PDB that contain the 4A/3B motif, whose conformations are identical in the presence or absence of a metal ion. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:Protein_Science:4 .

Chemical or electrochemical oxidation of a nitroxide radical leads to formation of an oxoammonium cation (OAC). OAC is able, in basic aqueous solution, of water oxidation with the formation of oxygen, while OAC reduces back to the initial radical.

Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca+2 , Mg+2 , and Mn+2 was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn+2 and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca+2 and Mg+2 complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several water molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of RMSD values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Protein_Science:3.

Copper oxide nanoparticles (CuO-NPs) have been suggested as effective catalysts to degrade many persistent organic contaminants. In parallel, CuO-NPs are considered toxic to soil microorganisms, plants and human cells, possibly because they induce oxidative stress and generation of reactive oxygen species (ROS). However, the mechanism of the catalytic process and the generated ROS are poorly understood. Here we discuss the reaction mechanism of CuO-NPs during the catalytic degradation of enrofloxacin – an antibiotic pharmaceutical used in this study as a representative persistent organic compound. The degradation of an aqueous solution of the enrofloxacin exposed to CuO-NPs and hydrogen peroxide was studied showing fast removal of the enrofloxacin at ambient conditionsns. ROS production was identified by electron spin resonance and a spin trapping technique. The distribution of the free radical species indicated production of a high percentage of superoxide (O2−.) radicals as well as hydroxyl radicals; this production is similar to the “radical production” activity of the superoxide dismutase (SOD) enzyme in the presence of hydrogen peroxide. This activity was also tested in the opposite direction, to examine if CuO-NPs show reactivity that potentially mimics the classical SOD enzymatic activity. The CuO-NPs were found to catalyze the dismutation of superoxide to hydrogen peroxide and oxygen in a set of laboratory experiments.

In this report, we present a method to characterize the kinetics of electron transfer across the bilayer of a unilamellar liposome composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The method utilizes synthetic phospholipids containing noninvasive nitroxide spin labels having the >N–O• moiety at well-defined distances from the outer surface of the liposome to serve as reporters for their local environment and, at the same time, permit measurement of the kinetics of electron transfer. We used 5-doxyl and 16-doxyl stearic acids. The paramagnetic >N–O• moiety is photo-oxidized to the corresponding diamagnetic oxoammonium cation by a ruthenium electron acceptor formed in the solution. Electron transfer is monitored by three independent spectroscopic methods: by both steady-state and time-resolved electron paramagnetic resonance and by optical spectroscopy. These techniques allowed us to differentiate between the electron transfer rates of nitroxides located in the outer leaflet of the phospholipid bilayer and of those located in the inner leaflet. Measurement of electron transfer rates as a function of temperature revealed a low-activation barrier (ΔG‡ ∼ 40 kJ/mol) that supports a tunneling mechanism.

In this review, we first survey the mechanisms underlying the chemical modification of amino acid residues in proteins by singlet oxygen elicited by photosensitizers. Singlet oxygen has the capacity to cause widespread chemical damage to cellular proteins. Its use in photodynamic therapy of tumors thus requires the development of methodologies for specific addressing of the photosensitizer to malignant cells while sparing normal tissue. We describe three targeting paradigms for achieving this objective. The first involves the use of a photosensitizer with a high affinity for its target protein; in this case, the photosensitizer is methylene blue for acetylcholinesterase. The second paradigm involves the use of the hydrophobic photosensitizer hypericin, which has the capacity to interact selectively with partially unfolded forms of proteins, including nascent species in rapidly dividing or virus-infected and cancer cells, acting preferentially at membrane interfaces. In this case, partially unfolded molten globule species of acetylcholinesterase serve as the model system. In the third paradigm, the photodynamic approach takes advantage of a general approach in ‘state-of-the-art’ chemotherapy, by coupling the photosensitizer emodin to a specific peptide hormone, GnRH, which recognizes malignant cells via specific GnRH receptors on their surface.

SIGNIFICANCE:Chemotherapy is currently the principal method for treating many malignancies. Thus, the development of improved antitumor drugs with enhanced efficacy and selectivity remains a high priority. Recent Advances: Anthracycline antibiotics (AAs), for example, doxorubicin, daunomycin, and mitomycin C, belong to an important family of antitumor agents widely used in chemotherapy. These compounds are all quinones. They are, thus, capable of being reduced by appropriate chemicals or reductases. One of their important properties is that under aerobic conditions their reduced forms undergo oxidation, with concomitant generation of reactive oxygen species (ROS), namely, superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals. The presence of metal ions is essential for the generation of ROS by AAs in biological systems.CRITICAL ISSUES:A fundamental shortcoming of the AAs is their high cardiotoxicity. We have proposed, and experimentally realized, a new type of quinones that is capable of coordinating metal ions. We have demonstrated in vitro that they can be reduced by electron transfer chains and glutathione with concomitant generation of ROS. They can also produce ROS under photo-excitation. The mechanisms of these reactions have been characterized by using nuclear magnetic resonance and electron paramagnetic resonance.FUTURE DIRECTIONS:To enhance their therapeutic effectiveness, and decrease cardiotoxicity and other side effects, we intend to conjugate the quinone chelators with monoclonal antibodies and peptide hormones that are specifically targeted to receptors on the cancer cell surface. Some such candidates have already been synthesized. An alternative approach for delivery of our compounds involves the use of specific peptide-based nanoparticles. In addition, our novel approach for treating malignancies is also suitable for photodynamic therapy. Antioxid. Redox Signal. 28, 1394-1403.

In order to limit the side effects associated with antitumor drugs such as doxorubicin, nanosized drug-delivery systems capable of selectively delivering and releasing the drug in the diseased tissue are required. We describe nanoparticles (NPs), self-assembled from a reduction responsive amphiphilic peptide, capable of entrapping high amounts of a redox active anticancer drug candidate and releasing it in presence of a reducing agent. This system shows a high entrapment efficiency with up to 15mg drug per gram of peptide (5.8mol-%). Treatment of the NPs with reducing agent results in the disassembly of the NPs and release of the drug molecules. A reduction in cell viability is observed at drug concentrations above 250nm in HEK293T and HeLa cell lines. This drug delivery system has potential for targeting tumor sites via the EPR effect while taking advantage of the increased reduction potential in tumor microenvironment.

Objectives: Alliinase is a pyridoxal-5’-phosphate (PLP)-dependent enzyme responsible for the production of diallyl thiosulfinate (allicin), the biologically active component of garlic, from alliin. The use of allicin for treatment of various diseases has been proposed but it is very unstable in the blood stream. This difficulty can be overcome by administration of alliin, together with a conjugate of alliinase directed towards the target cells. This, in turn requires a stable and active form of the enzyme. In this study we evaluate the stability of alliinase itself, in the presence and absence of osmolytes, as well as that of its catalytically active complex with a mannose-specific lectin, ASAI (Allium sativum agglutinin I), also presents in garlic. Methods: Alliinase, and ASAI were both purified from garlic cloves. Thermal stability of alliinase itself, and of its complexes with PLP and ASAI, in the presence and absence of osmolytes, was analyzed by monitoring enzymic activity, and using DSC (differential scanning calorimetry). Key findings: PLP exerts only a minor influence on alliinase structure and stablity. But both osmolytes and ASAI stabilize the enzyme considerably. Conclusions: The principle finding is that ASAI greatly stabilizes alliinase. Thus, the lectin-enzyme complex, which can be lyophilized and stored until used, provides an effective formulation of alliinase for generation of allicin from alliin in vivo.

Xanthorhodopsin (xR) is a member of the retinal protein family and acts as a proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber. In addition to the retinal chromophore, xR contains a carotenoid, which acts as a light-harvesting antenna as it transfers 40% of the quanta it absorbs to the retinal. Our previous studies have shown that the CD and absorption spectra of xR are dramatically affected due to the protonation of two different residues. It is still unclear whether xR can bind cations. Electron paramagnetic resonance (EPR) spectroscopy used in the present study revealed that xR can bind divalent cations, such as Mn2+ and Ca2+, to deionized xR (DI-xR). We also demonstrate that xR can bind 1 equiv of Mn2+ to a high-affinity binding site followed by binding of ∼40 equiv in cooperative manner and ∼100 equiv of Mn2+ that are weakly bound. SQUID magnetic studies suggest that the high cooperative binding of Mn2+ cations to xR is due to the formation of Mn2+ clusters. Our data demonstrate that Ca2+ cations bind to DI-xR with a lower affinity than Mn2+, supporting the assumption that binding of Mn2+ occurs through cluster formation, because Ca2+ cations cannot form clusters in contrast to Mn2+.

Abstract The role of metal ions in the mechanism of light-stimulated redox activity of potential anticancer agent 2-phenyl-4-(butylamino)naphtha[2,3-h]quinoline-7,12-dione (Qc) has been studied by CIDNP (chemically induced dynamic nuclear polarization) and EPR methods. The photo-induced oxidation of NADH and its synthetic analog – substituted dihydropyridine (DHP) – by quinone Qc was used as a model. The Qc capability of producing chelating complexes with divalent metal ions of Fe, Zn and Ca was studied quantitatively by optical absorption spectroscopy. A significant decrease of electrochemical reduction potential of Qc (ΔE=0.4−0.6 eV for ACN and ACN/PBS solutions) in chelating complexes and in protonated form of Qc was observed. A pronounced increase in efficiency of DHP oxidation in chelating complexes with Zn2+ and Ca2+ ions compared with free Qc was demonstrated. The yields of free radicals, including reactive oxygen species (ROS) and reaction products, were a few times higher than those in the absence of metal ions. Application of such chelating compounds to enhance ROS generation looks very promising for anti-cancer therapy, including the photodynamic therapy.

Structure-based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ∼3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π-cation interactions with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design.

Two pH-sensitive stable nitroxide radicals are characterized by EPR spectroscopy and cyclic voltammetry in aqueous solution at different pH values. The rates of the photochemically-induced electron transfer reactions from the protonated and deprotonated forms of the radicals to a ruthenium complex are determined.

A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl(-) pump. It was recently demonstrated that the Natronomonas pharaonis halorhodopsin-overproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin, which binds to crevices between adjacent protein subunits. It is established that halorhodopsin has several chloride binding sites, with binding site I, located in the retinal protonated Schiff base vicinity, affecting retinal absorption. However, it remained unclear whether cations also bind to this protein. Our electron paramagnetic resonance spectroscopy examination of cation binding to the halorhodopsin mutant KM-1 reveals that divalent cations like Mn(2+) and Ca(2+) bind to the protein. Halorhodopsin has a high affinity for Mn(2+) ions, which bind initially to several strong binding sites and then to binding sites that exhibit positive cooperativity. The binding behavior is pH-dependent, and its strength is influenced by the nature of counterions. Furthermore, the binding strength of Mn(2+) ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn(2+) ions, as well as Cl(-) ions, first occupy binding sites other than site I. The observed synergetic effect between cation and anion binding suggests that while Cl(-) anions bind to halorhodopsin at low concentrations, the occupancy of site I requires a high concentration.

A unique mode of molecular oxygen activation, involving metal-ligand cooperation, is described. Ir pincer complexes [((t)BuPNP)Ir(R)] (R = C6H5 (1), CH2COCH3 (2)) react with O2 to form the dearomatized hydroxo complexes [((t)BuPNP*)Ir(R)(OH)] ((t)BuPNP* = deprotonated (t)BuPNP ligand), in a process which utilizes both O-atoms. Experimental evidence, including NMR, EPR, and mass analyses, indicates a binuclear mechanism involving an O-atom transfer by a peroxo intermediate.

The production of singlet oxygen is typically associated with inefficient dissipation of photosynthetic energy or can arise from light reactions as a result of accumulation of chlorophyll precursors as observed in fluorescent (flu)-like mutants. Such photodynamic production of singlet oxygen is thought to be involved in stress signaling and programmed cell death. Here we show that transcriptomes of multiple stresses, whether from light or dark treatments, were correlated with the transcriptome of the flu mutant. A core gene set of 118 genes, common to singlet oxygen, biotic and abiotic stresses was defined and confirmed to be activated photodynamically by the photosensitizer Rose Bengal. In addition, induction of the core gene set by abiotic and biotic selected stresses was shown to occur in the dark and in nonphotosynthetic tissue. Furthermore, when subjected to various biotic and abiotic stresses in the dark, the singlet oxygen-specific probe Singlet Oxygen Sensor Green detected rapid production of singlet oxygen in the Arabidopsis (Arabidopsis thaliana) root. Subcellular localization of Singlet Oxygen Sensor Green fluorescence showed its accumulation in mitochondria, peroxisomes, and the nucleus, suggesting several compartments as the possible origins or targets for singlet oxygen. Collectively, the results show that singlet oxygen can be produced by multiple stress pathways and can emanate from compartments other than the chloroplast in a light-independent manner. The results imply that the role of singlet oxygen in plant stress regulation and response is more ubiquitous than previously thought.

High-valent oxo compounds of transition metals are often implicated as active species in oxygenation of hydrocarbons through carbon-hydrogen bond activation or oxygen transfer and also in water oxidation. Recently, several examples of cobalt-catalyzed water oxidation have been reported, and cobalt(IV) species have been suggested as active intermediates. A reactive species, formally a dicobalt(IV)-μ-oxo polyoxometalate compound [(α2-P2W17O61Co)2O](14-), [(POMCo)2O], has now been isolated and characterized by the oxidation of a monomeric [α2-P2W17O61Co(II)(H2O)](8-), [POMCo(II)H2O], with ozone in water. The crystal structure shows a nearly linear Co-O-Co moiety with a Co-O bond length of ∼1.77 Å. In aqueous solution [(POMCo)2O] was identified by (31)P NMR, Raman, and UV-vis spectroscopy. Reactivity studies showed that [(POMCo)2O]2O] is an active compound for the oxidation of H2O to O2, direct oxygen transfer to water-soluble sulfoxides and phosphines, indirect epoxidation of alkenes via a Mn porphyrin, and the selective oxidation of alcohols by carbon-hydrogen bond activation. The latter appears to occur via a hydrogen atom transfer mechanism. Density functional and CASSCF calculations strongly indicate that the electronic structure of [(POMCo)2O]2O] is best defined as a compound having two cobalt(III) atoms with two oxidized oxygen atoms.