Chelation of hypocrellin B with zinc ions with electron paramagnetic resonance (EPR) evidence of the photodynamic activity of the resulting chelate

Hypocrellin B (HB), a perylenequinone derivative, is an efficient phototherapeutic agent. The chelation of HB with Zinc ions (Zn²⁺) results in a metal chelate (Zn-HB) which exhibits considerable absorption (λ_max = 612nm) in the phototherapeutic window. The structure of this chelate has been characterized by UV-Vis, IR and mass spectra. The redox potentials of the Zn-HB chelate were E_ox = +1.1V (vs. SCE) and E_re = -0.7V (vs. SCE) as measured using the circle volt curve. The quantum yield of singlet oxygen generated by the Zn-HB chelate was 0.86, which both the electron spin trap (EPR) method and the chemical trap method show to be about 0.1 higher than that of its parent compound HB. In irradiated oxygen-saturated solutions of Zn-HB chelate, superoxide radical anions and hydroxyl radicals were detected by EPR spectroscopy using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping agent.     

A new Co(III)-hypocrellin B complex with enhanced photonuclease activity

Hypocrellin B (HB), a naturally occurring photosensitizer, has been extensively and intensively studied as a promising photodynamic therapy (PDT) agent. In this work, a new Co(III) complex [Co₂(HB)(tmp)₄]⁴⁺ (tmp = 3,4,7,8-tetramethyl-1,10-phenanthroline) was designed and synthesized with HB as bridging ligand and tmp as terminal ligand. [Co₂HB(tmp)₄]⁴⁺ exhibits improved water solubility, enhanced absorptivity in the phototherapeutic window, increased binding affinity and DNA photocleavage capability toward dsDNA with respect to HB. The photodynamic activity of [Co₂(HB)(tmp)₄]⁴⁺ stems from its ¹O₂ photosensitization ability, in sharp contrast to [Cu₂(HB)(tmp)₂]²⁺ which relies on superoxide anion radical (O₂^-) and hydroxyl radical (·OH) to photocleave DNA, though the both complexes possess similar electrochemical properties. The remarkable difference between the photodynamic mechanisms of [Co₂(HB)(tmp)₄]⁴⁺ and [Cu₂(HB)(tmp)₂]²⁺ was discussed in detail.     

Photo-induced electron transfer between hypocrellins and nano-sized semiconductor CdS —EPR study on the kinetics of photosensitized reduction in HA-CdS and HB-CdS systems

Both HA-CdS and HB-CdS (Hys-CdS, Hys represents HA, HB) complex systems were established according to the dynamics of heterogeneous electron-transfer process m = E_{S^* /S⁺} - E < 0\mu = E_{S^* /S^ + } - E< 0 . In these systems, the electron transferring from¹Hys* to conduction band of CdS is feasible. Determined from the fluorescence quenching, the apparent association constants (K_app) between Hypocrellin A (HA), Hypocrellin B. (HB) and CdS sol. were about 940 (mol/L)⁻¹, 934 (mol/L)⁻¹, respectively. Fluorescence lifetime measurements gave the rate constant for the electron transfer process from¹HA*,¹HB* into conduction band of CdS semiconductor as 5.16 × 10⁹ s⁻¹, 5.10 × 10⁹ s⁻¹, respectively. TEMPO (2,2,6,6-tetramethy-1-piperdinyloxy), a stable nitroxide radical, was used in the kinetic study of the reduction reaction taking place on the surface of a CdS colloidal semiconductor, kinetics equation of the reaction was determined with the electron paramagnetic resonance (EPR) method, and the reaction order of TEMPO is zero. When Hys were added, the rate of EPR increased greatly. By comparing rate constants, the Hys-CdS systems were revealed to be about 350 times more efficient than CdS sol. alone in the photoreduction of TEMPO under visible light. It suggests that Hys can be used as efficient sensitizers of a colloidal semiconductor in the application of solar energy.     

Potential‐resolved electrochemiluminescence of Ru(bpy)32+/C2O42− system on gold electrode

The electrogenerated chemiluminescence of Ru(bpy)₃²⁺/C₂O₄^2? system on a pre‐polarized Au electrode was studied using a potential‐resolved electrochemiluminescence (PRECL) method. Two anodic ECL peaks were observed at 1.22 V (vs. SCE) (EP1), 1.41 V (vs. SCE) (EP2), respectively. The effects of the concentration of oxalate and Ru(bpy)₃²⁺, adsorbed sulphur, CO₂, O₂, pH of the solution and pretreatment of the Au electrode on the two PRECL peaks were examined. The surface state of the pre‐oxidized gold electrode was also studied using the X‐ray photoelectron spectroscopy (XPS) technique. Moreover, comparative studies on i–E and I–E curves were carried out and a possible mechanism involving both the catalytic and the direct electro‐oxidation pathways was proposed for the ECL of Ru(bpy)₃²⁺/C₂O₄^2? system. EP1 is attributed to the Ru(bpy)₃^2/3+ reaction catalysed by C₂O₄^2? to generate Ru(bpy)₃^2+*. EP2 is likely because C₂O₄^2? was oxidized at the electrode to form CO₂^?·, followed by reaction with Ru(bpy)₃³⁺ to generate Ru(bpy)₃^2+*. Copyright © 2002 John Wiley & Sons, Ltd.     

Redox chemistry of biological tungsten: an EPR study of the aldehyde oxidoreductase from Pyrococcus furiosus

 Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins. A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]¹⁺ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E _m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the cofactor. Received: 18 December 1995 / Accepted: 26 March 1996     

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis

Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe₄S₄]^2+,+ cluster (E _m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and W(V)/W(VI) states at physiologically relevant potentials (E _m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na₂S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E _m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E _m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR. Received: 9 September 1999 / Accepted: 17 February 2000     

Evidence of in vivo Free Radical Generation by Spin Trapping with α-Phenyl N-Tert-Butyl Nitrone During Ischemia/Reperfusion in Rabbit Kidneys

By using α-phenyl N-tert-butyl nitrone (PBN) as spin trap molecule and the electron paramagnetic resonance (EPR) technique, we obtained the first direct evidence of in vivo intervention of free radicals during an ischemia (50 minutes) reperfusion phenomenon in kidney of an intact rabbit.

An EPR signal (triplet of doublets) characterized by coupling constants a_N = 14.75–15 G and a^H_s = 2.5–3 G was detected in blood samples. The signal was consistent with a nitroxyl-radical adduct resulting from the spin trapping by PBN of either oxygen-or carbon-centered radicals. Control experiments indicated that the EPR signal was not due to a toxic effect of the spin trap molecule.     

Development of methods for functionalization of screen printed electrodes with biocompatible organic-inorganic hybrid nanocomposites for biosensing applications

New types of organic-inorganic hybrid nanocomposites based on nanosized titanium oxide(IV) (TiO₂, particle size <100 nm) and carbon nanotubes (CNT, outer diameter of 10–15 nm, inner diameter of 2–6 nm, and length of 0.1–10 μm) and phosphatidylcholine were elaborated for improvement of analytical characteristics of screen printed electrodes. These nanomaterials were employed as an interface for immobilization of skeletal myoglobin. Electroanalytical and electrokinetic behavior of myoglobin on such interfaces was characterized with cyclic voltammetry (CV) and square wave voltammetry (SWV). Direct unmediated electron transfer between heme of immobilized myoglobin and electrodes modified with titanium oxide or carbon nanotubes was registered. The midpoint (redox) potential of the myoglobin Fe³⁺/Fe²⁺ E _1/2 = ?0.263 V for electrodes modified with CNT and E _1/2 = ?0.468 V for electrodes modified with TiO₂ was observed (vs. Ag/AgCl reference electrode).     

Metal chelates in the storage and transport of neurotransmitters: formation of mixed ligand chelates of Mg 2+ -ATP with biogenic amines

Abstract— Quantitative studies on the interactions of adenosine-triphosphate and several biogenic amines with magnesium ion have been carried out in an attempt to correlate the thermodynamic stabilities of the metal-binding of the amines with the in vivo affinities of the amines for granule-binding. Equilibrium data indicate that in each of the ternary chelate systems (viz. Mg²⁺-ATP-amine), the predominant reaction in the pH range 3.0–7.0 is the formation of a magnesium-ATP chelate with a stability constant, log K_ML=3.22 ± 0.02. Each of the biogenic amines coordinates with Mg²⁺-ATP system in the pH range 7.0–10.5 to form the mixed ligand chelate (or ternary chelate), Mg²⁺-ATP-amine(1:1:1). The stability constants for the binding of the amines with Mg²⁺-ATP are: (i) norepinephrine (NE) = 2.34 ± 0.32; (ii) epinephrine (E) = 2.95 ± 0.08; (iii) dopamine (DA) = 3.05 ± 0.06; (iv) octopamine (OA) = 1.93 ± 0.12; (v) 6-hydroxydopamine (6-HDA) = 2.42 ± 0.14; (vi) 3-methoxynorephedrine (MeN) =2.76 ± 0.09; (vii) amphetamine (AA) =2.09 ± 0.05; (viii) tyramine (TA) = 2.60 ± 0.04; (ix) phenylethylamine (PEA) = 0. A general correlation is indicated between the stability constants (binding strengths) of the amine chelates and the metal-binding functionalities of the amines on the one hand and their vesicular binding characteristics in in vivo systems on the other (Carlsson and Waldeck , 1966). The Mg²⁺-ATP-dependant amine storage mechanism of KIRSHNER (1962a;b) and Carlsson , Hillårp and Waldeck (1963) is discussed both in the light of the data on metal chelate stability and of a significant modification of metal coordination hypothesis.     

Photobiochemistry of folates: A photochemical reduction of folic acid

Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310–390 nm, I = 0.4 W m⁻²) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E′_o = +0.40 V), while the presence of stronger reductants—NADH (E′_o = −0.32 V) and boron hydride (E′_o = −0.48 V)—induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid.     

Pilot scale production of poly(3-hydroxybutyrate-co-3-hydroxy-valerate) by fed-batch culture of recombinantEscherichia coli

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)], by fed-batch culture of recombinantEscherichia coli harboring a plasmid containing theAlcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes, was examined in two pilot-scale fermentors with air supply only. In a 30 L fermentor having aK _La value of 0.11 s⁻¹, the final P(3HB/V) concentration and the P(3HB/V) content obtained were 29.6 g/L and 70.1 wt%, respectively, giving a productivity of 1.37 g P(3HB/V)/L-h. In a 300 L fermentor having aK _La of 0.03 s⁻¹, the P(3HB/V) concentration and the P(3HB/V) content were 20.4 g/L and 69 wt%, respectively, giving a productivity of 1.06 g P(3HB/V)/L-h. These results suggest that economical production of P(3HB/V) is possible by fed-batch culture of recombinantE. coli in a large-scale fermentor having lowK _La value.     

Spin Trapping of Ibuprofen Radicals: Evidence That Ibuprofen is a Hydroxyl Radical Scavenger

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2–(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vim. Ibuprofen (IBU; 0.1–50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5–2.0mM). and either 5.5-dimethyl-l-pyrroline-N-oxide (DMPO; 40mM) or a-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (l mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (a_N = 16.1G, a^H_β = 24.0G) and PBN (a_N = 15.7G. a^H_β = 4.4G and a_N = 17.0G, a^H_β = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (l mM), hydrogen peroxide (lmM), PBN (48mM), and increasing IBU concentrations. Therefore. we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hydroxyl radicals would be generated, less IBU radicals formed and the signal height of IBU radicals trapped by PBN would have decreased. However, these data do not fully exclude the possiblity that IBU may, to some extent. also chelate iron. Scavenging of hydroxyl radicals may be one of the mechanisms responsible for the beneficial action of IBU during the management of several rheumatic diseases. However, the IBU radicals produced when IBU scavenges hydroxyl radicals are reactive. and may be associated with the reported toxicity of this therapeutic agent.     

Sources of divalent sulfur allow recovery of the Fnr [4Fe-4S]<Superscript>2+</Superscript> center in <Emphasis Type="Italic">Escherichia coli</Emphasis> incubated with nitric oxide donors

Dinitrosyl iron complexes (DNICs) with various thiol ligands, the known donors of nitric oxide, markedly inhibited aidB gene expression in E. coli cells by destroying the [4Fe-4S]²⁺ center of its regulator protein Fnr. Therewith, the cells accumulated DNICs in the protein-bound form, identified by the EPR signal with g _⊥ = 2.04 and g _‖ = 2.014. Subsequent addition of sulfur sources L-cysteine or N-acetylcysteine, DTT as well as Na₂S to the DNIC-treated cells significantly restored the reporter gene expression. Simultaneously, the above-specified EPR signal was partly or completely replaced with a narrower signal (g _⊥ = 2.032, g _‖ = 2.02) identical to that of DNICs with persulfide (R-S-S⁻) ligands, which result from interaction of S²⁻ with thiols; inorganic sulfide proved to be the most efficient agent. These data corroborate the central role of S²⁻ in recovery of the protein [4Fe-4S] center disrupted by the NO donors.     

Stimulation and oxidative catalytic inactivation of thermolysin by copper•Cys-Gly-His-Lys

[Cu²⁺•Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 μM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 μM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 μM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu²⁺–amino-terminal copper and nickel binding motif (λ _max ∼ 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu²⁺ charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (ΔE = 92 mV) for the Cu^3+/2+ redox couple obtained for [Cu²⁺•Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu²⁺•Lys-Gly-His-Lys]⁺ (0.84 V, ΔE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu²⁺•Lys-Gly-His-Lys]⁺ homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O₂) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k _obs = 7.7 × 10⁻² min⁻¹, yielding a second-order rate constant of (7.7 ± 0.9) × 10⁴ M⁻¹ min⁻¹. Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.     

Redox chemistry of tungsten and iron–sulfur prosthetic groups in Pyrococcus furiosus formaldehyde ferredoxin oxidoreductase

Formaldehyde oxidoreductase (FOR) is one of the tungstopterin iron–sulfur enzymes of the five-membered family of aldehyde oxidoreductases in the hyperthermophilic archaeon Pyrococcus furiosus. In dye-mediated equilibrium redox titrations, the tungsten in active P. furiosus FOR is a two-electron acceptor, W(VI/IV). The intermediate, paramagnetic W(V) state can be trapped only by reduction with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe–4S]^(2+;+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Electron paramagnetic resonance (EPR) spectroscopy reveals a single “low-potential” W(V) spectrum with g _xyz values 1.847, 1.898, and 1.972, and a [4Fe–4S]⁺ cubane in a spin mixture of S = 1/2 (10%) and S = 3/2 (90%) of intermediate rhombicity (E/D = 0.21, g _real = 1.91). The development of this intermediate in vitro is slow even at elevated temperature and with a nominal 50:1 excess of substrate over enzyme presumably owing to the very unfavorable hydration equilibrium of the formaldehyde/methylene glycol couple with K _D ≈ 10³. Rapid intermediate formation of enzyme at concentrations suitable for EPR spectroscopy (200 μM) is only obtained with extremely high nominal substrate concentration (1 M formaldehyde) and is followed by a slower phase of denaturation. The premise that the free formaldehyde, and not the methylene glycol, is the enzyme’s substrate implies that K _M for formaldehyde is 3 orders of magnitude less that the previously reported value.     

Studies of the function of the membrane-bound iron-sulfur centers of the photosynthetic bacterium Chromatium vinosum

Chromatophores from the photosynthetic bacterium, Chromatium vinosum, have been prepared which photoreduce NAD⁺ with either succinate or reduced dichlorophenolindophenol as electron donors. NAD⁺ reduction is inhibited by uncouplers as well as inhibitors of cyclic photophosphorylation. These chromatophores contain several bound iron-sulfur centers which have been detected by low-temperature EPR spectroscopy. One center, having a g 2.01 EPR signal in the oxidized state, has E_m7.5 = +50 mV and is partially reduced by succinate in the dark. Three iron-sulfur centers having g 1.93 EPR signals have been resolved by redox titration, and the E_m7.5 values of these centers are ?50, ?175 and ?250 mV, respectively. Studies of the involvement of these centers in electron transfer from donors to NAD⁺ have indicated that the center with E_m = ?50 mV is succinate reducible in the dark and appears to be analogous to center S-1 of succinic dehydrogenase in other systems. An additional g 1.93 iron-sulfur center can be photoreduced in the presence of electron donors and this reduction is inhibited by uncouplers. The possible role of the two low-potential iron-sulfur centers in relation to the dehydrogenases functioning in NAD⁺ reduction is considered.     

Peroxyl radical is produced upon the interaction of hypochlorite with tert-butyl hydroperoxide

As we reported previously, hypochlorite interacting with organic hydroperoxides causes their decomposition ((1995) Biochemistry (Moscow), 60, 1079-1086). This interaction was supposed to be a free-radical process and serve as a source of free radicals initiating lipid peroxidation (LP). The present study is the first attempt to detect and identify free radicals produced in the reaction of hypochlorite with tert-butyl hydroperoxide, (CH₃)₃COOH, which we have used as an example of organic hydroperoxides. We have used a direct method for free radical detection, EPR of spin trapping, and the following spin traps: N-tert-butyl--phenylnitrone (PBN) and -(4-pyridyl-1-oxyl)-N-tert-butylnitrone (4-POBN). When hypochlorite was added to (CH₃)₃COOH in the presence of a spin trap, an EPR spectrum appeared representing a superposition of two signals. One of them belonged to a spin adduct formed as a result of direct interaction of hypochlorite with the spin trap (hyperfine splitting constants were: _H H = 0.148 mT; a_N = 1.537 mT; and H_PP = 0.042 mT for 4-POBN and _H = 0.190 mT; a_N = 1.558 mT; and H_PP = 0.074 mT for PBN). The other signal was produced by hypochlorite interactions with (CH3)3COOH itself (hyperfine splitting constants were: _H = 0.233 mT; aN = 1.484 mT; H_PP = 0.063 mT and _H = 0.360 mT; a_N = 1.547 mT; H_PP = 0.063 mT for 4-POBN and PBN, respectively). Comparison of spectral characteristics of this spin adduct with those of tert-butoxyl or tert-butyl peroxyl radicals produced in known reactions of (CH₃)₃COOH with Fe²⁺ and Ce⁴⁺, respectively, showed that the radical (CH₃)₃COO. is produced from the interaction of hypochlorite with (CH₃)₃COOH^. Like Ce⁴⁺ but not Fe²⁺, hypochlorite addition to (CH₃)₃COOH was accompanied by a bright flash of chemiluminescence characteristic of the reactions in which peroxyl radicals are produced. Thus, all these results suggest peroxyl radical production in the reaction of hypochlorite with hydroperoxide. This reaction is one of the most possible ways for the initiation of free-radical LP that occurs in vivo, when hypochlorite interacts with unsaturated lipids comprising natural protein–lipid complexes, such as lipoproteins and biological membranes.     

Analysis of the electrochemistry of hemes with Ems spanning 800 mV

The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of E_ms with a single type of redox cofactor. Proteins containing 141 unique hemes of a‐, b‐, and c‐type, with bis‐His, His‐Met, and aquo‐His ligation were calculated using Multi‐Conformation Continuum Electrostatics (MCCE). The experimental E_ms range over 800 mV from ?350 mV in cytochrome c₃ to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the protein backbone, and sidechains. MCCE calculated E_ms are in good agreement with measured values. Using no free parameters the slope of the line comparing calculated and experimental E_ms is 0.73 (R² = 0.90), showing the method accounts for 73% of the observed E_m range. Adding a +160 mV correction to the His‐Met c‐type hemes yields a slope of 0.97 (R² = 0.93). With the correction 65% of the hemes have an absolute error smaller than 60 mV and 92% are within 120 mV. The overview of heme proteins with known structures and E_ms shows both the lowest and highest potential hemes are c‐type, whereas the b‐type hemes are found in the middle E_m range. In solution, bis‐His ligation lowers the E_m by ≈205 mV relative to hemes with His‐Met ligands. The bis‐His, aquo‐His, and His‐Met ligated b‐type hemes all cluster about E_ms which are ≈200 mV more positive in protein than in water. In contrast, the low potential bis‐His c‐type hemes are shifted little from in solution, whereas the high potential His‐Met c‐type hemes are raised by ≈300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. For example, the loss of solvation (reaction field) energy, which raises the E_m, has been suggested to be a major factor in tuning in situ E_ms. However, the calculated solvation energy vs. experimental E_m shows a slope of 0.2 and R² of 0.5 thus correlates weakly with E_ms. All other individual interactions show even less correlation with E_m. However the sum of these terms does reproduce the range of observed E_ms. Therefore, different proteins use different aspects of their structures to modulate the in situ heme electrochemistry. This study also shows that the calculated E_ms are relatively insensitive to different heme partial charges and to the protein dielectric constant used in the simulation. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Direct Nanoscale Characterization of Deep Levels in AgCuInGaSe2 Using Electron Energy‐Loss Spectroscopy in the Scanning Transmission Electron Microscope

A new experimental framework for the characterization of defects in semiconductors is demonstrated. Through the direct, energy‐resolved correlation of three analytical techniques spanning six orders of magnitude in spatial resolution, a critical mid‐bandgap electronic trap level (E_V + 0.56 eV) within Ag_0.2Cu_0.8In_1?xGa_xSe₂ is traced to its nanoscale physical location and chemical source. This is achieved through a stepwise, site‐specific correlated characterization workflow consisting of device‐scale (≈1 mm²) deep level transient spectroscopy (DLTS) to survey the traps present, scanning probe–based DLTS (scanning‐DLTS) for mesoscale‐resolved (hundreds of nanometers) mapping of the target trap state's spatial distribution, and scanning transmission electron microscope based electron energy‐loss spectroscopy (STEM‐EELS) and X‐ray energy‐dispersive spectroscopy for nanoscale energy‐, structure, and chemical‐resolved investigation of the defect source. This first demonstration of the direct observation of sub‐bandgap defect levels via STEM‐EELS, combined with the DLTS methods, provides strong evidence that the long‐suspected Cu_In/Ga substitutional defects are indeed the most likely source of the E_V + 0.56 eV trap state and serves as a key example of this approach for the fundamental identification of defects within semiconductors, in general.     

Interaction of tert -butyl Hydroperoxide with Hypochlorous Acid. A Spin Trapping and Chemiluminescence Study

The formation of radical species during the reaction of tert-butyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap &#102 -(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (a_N = 1.537mT, a^&#103_H = 0.148mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (a_N = 1.484mT, a^&#103_H = 0.233mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl- &#102 -phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe²⁺ or Ce⁴⁺ in the presence of POBN. Using Fe²⁺ a spin adduct with a _N= 1.633mT and a^&#103_H = 0.276mT was observed. The major spin adduct formed with Ce⁴⁺ was characterised by α_N = 1.480mT and a^&#103_H = 0.233mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce⁴⁺. The addition of Fe²⁺ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce⁴⁺ the same spin adduct and the same luminescence profile. Because Ce⁴⁺ is known to oxidise organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.