One-electron reduction of halothane and formation of halide ions in aqueous solutions

Formation of Br^? and, under certain conditions also F^? ions has been observed in the radiation chemically induced one-electron reduction of the anesthetic halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in aqueous solutions. The initial step is the release of Br^? and formation of the 2-chloro-1,1,1-trifluoroethyl radical. The latter can react via competing pathways including H-atom abstraction, addition of molecular oxygen and further reduction by an antioxidant. All of these three competitive routes lead to different product patterns. High yields of F^? ions are observed under anaerobic conditions in the presence of antioxidants such as ascorbate, propylgallate, etc. The fluoride elimination is strongly pH-dependent and seems to occur in various steps after initiation through reduction of the (CF₃CHCl) radical. The implication for biochemical studies on the metabolism of halothane under different oxygen concentrations is discussed.     

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments

The one-electron reduction potentials of the radical cations of five dietary carotenoids (β-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.     

One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

One-electron reduction of chromate by NADPH-dependent glutathione reductase

Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (.OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the .OH radical and Cr(V) formation, indicating that the O2- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the .OH radical formation, indicating the involvement of hydrogen peroxide (H2O2) in the reaction mechanism. Addition of H2O2 suppresses Cr(V) and enhances the .OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate .OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.     

One-electron reduction of chloroperoxidase by radiolytically generated electrons

Upon irradiation of aqueous ethylene glycol/water solutions of native chloroperoxidase (CPO) with 60Co-gamma rays at 77K one observes the one-electron reduction of the enzyme active site by radiolytically generated thermolyzed electrons. In the present study the first absorption spectrum of a low-spin ferrous form of CPO is reported which has peaks at 438, 532 and 563 nm, similar to those observed previously for cytochrome P-450. All previously described ferrous forms of CPO are high spin. In order to observe the final results of the CPO reaction with electrons, the spectral changes of native enzyme after room temperature-gamma-irradiation have also been investigated. Evidence of changes is also presented probably connected with disruption of the tertiary structure of enzyme, correlated with decrease of enzyme activity.     

One-electron reduction of the oxy form of cobalt-substituted hemoproteins

The reduction of oxy forms in cobalt-substituted hemoproteins by the hydrated electron (e(aq)-) was investigated by pulse radiolysis. The hydrated electron (e(aq)-) reacted with the oxy form of cobalt horseradish peroxidase (CoHRP) to form CoHRP. On the other hand, the initial product observed in the reaction of the oxy form of cobalt myoglobin (CoMb) with e(aq)- is neither CoMb nor Co3+ Mb. Subsequently, the product was found to convert to another form, the irreversible change in the porphyrin. In contrast to e(aq)-, both oxy forms of CoMb and CoHRP were reduced by various electron donors to form the cobaltic forms.     

Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine

Abstract

Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (> 90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.     

One-electron reduction of adriamycin: Properties of the semiquinone

Pulse radiolysis of aqueous solutions containing adriamycin and redox indicators of known one-electron reduction potential (E¹) shows that its E¹ at pH 7 is ?328 mV (vs NHE). The variation E¹ with pH in the range 6–12 shows that the net charge on the semiquinone at pH 7 is zero. As well as the pK_a values of 2.9 and ≥ 14 established independently, the semiquinone has a pK_a close to 9.2. The new data enable the structure and likely reactivity of the semiquinone to be specified.     

One-electron reduction of flavodoxin. A fast kinetic study.

The reaction kinetics of fully oxidized flavodoxin from Clostridium MP with the hydrated electron have been investigated by the pulse radiolysis method. Four spectrally distinct processes have been observed with the ultimate formation of the singly reduced flavin form of the protein. The last two species obtained in the reaction sequence are spectrally similar, and are connected through a reaction which is first order. It is proposed that this reaction involves a protein conformational alteration.     

One-electron reduction of the oxyform of 2,4-diacetyldeuterocytochrome P-450cam

The reaction of the hydrated electron with a ferrous oxygenated form of modified cytochrome P-450cam, containing 2,4-diacetyldeuteroheme, was investigated by the use of pulse radiolysis. The ferrous oxygenated form of this enzyme was reduced by hydrated electrons to form the product, which exhibits absorption maximum at 470 and 370 nm. From the spectrum obtained, the oxidation state of the product is discussed in relation to the higher oxidation states of chloroperoxidase.     

One-electron oxidation and reduction of glycosaminoglycan chloramides: A kinetic study

Hypochlorous acid and its acid–base counterpart, hypochlorite ions, produced under inflammatory conditions, may produce chloramides of glycosaminoglycans, these being significant components of the extracellular matrix (ECM). This may occur through the binding of myeloperoxidase directly to the glycosaminoglycans. The N–Cl group in the chloramides is a potential selective target for both reducing and oxidizing radicals, leading possibly to more efficient and damaging fragmentation of these biopolymers relative to the parent glycosaminoglycans. In this study, the fast reaction techniques of pulse radiolysis and nanosecond laser flash photolysis have been used to generate both oxidizing and reducing radicals to react with the chloramides of hyaluronan (HACl) and heparin (HepCl). The strong reducing formate radicals and hydrated electrons were found to react rapidly with both HACl and HepCl with rate constants of 1–1.7×10⁸ and 0.7–1.2×10⁸ M⁻¹ s⁻¹ for formate radicals and 2.2×10⁹ and 7.2×10⁸ M⁻¹ s⁻¹ for hydrated electrons, respectively. The spectral characteristics of the products of these reactions were identical and were consistent with initial attack at the N–Cl groups, followed by elimination of chloride ions to produce nitrogen-centered radicals, which rearrange subsequently and rapidly to produce C-2 radicals on the glucosamine moiety, supporting an earlier EPR study by M.D. Rees et al. (J. Am. Chem. Soc. 125: 13719–13733; 2003). The oxidizing hydroxyl radicals also reacted rapidly with HACl and HepCl with rate constants of 2.2×10⁸ and 1.6×10⁸ M⁻¹ s⁻¹, with no evidence from these data for any degree of selective attack on the N–Cl group relative to the N–H groups and other sites of attack. The carbonate anion radicals were much slower with HACl and HepCl than hydroxyl radicals (1.0×10⁵ and 8.0×10⁴ M⁻¹ s⁻¹, respectively) but significantly faster than with the parent molecules (3.5×10⁴ and 5.0×10⁴ M⁻¹ s⁻¹, respectively). These findings suggest that these potential in vivo radicals may react in a site-specific manner with the N–Cl group in the glycosaminoglycan chloramides of the ECM, possibly to produce more efficient fragmentation. This is the first study therefore to conclusively demonstrate that reducing radicals react rapidly with glycosaminoglycan chloramides in a site-specific attack at the N–Cl group, probably to produce a 100% efficient biopolymer fragmentation process. Although less reactive, carbonate radicals, which may be produced in vivo via reactions of peroxynitrite with serum levels of carbon dioxide, also appear to react in a highly site-specific manner at the N–Cl group. It is not yet known if such site-specific attacks by this important in vivo species lead to a more efficient fragmentation of the biopolymers than would be expected for attack by the stronger oxidizing species, the hydroxyl radical. It is clear, however, that the N–Cl group formed under inflammatory conditions in the extracellular matrix does present a more likely target for both reactive oxygen species and reducing species than the N–H groups in the parent glycosaminoglycans.     

One-electron reduction of quinones by the neuronal nitric-oxide synthase reductase domain

Flavin electron transferases can catalyze one- or two-electron reduction of quinones including bioreductive antitumor quinones. The recombinant neuronal nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FMN prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADPH-oxidation in the presence of the model quinone compound menadione (MD), including antitumor mitomycin C (Mit C) and adriamycin (Adr). Calcium/calmodulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-mediated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxidoreductase (QR), but Mit C- and Adr-mediated NADPH oxidations were not. In anaerobic conditions, cytochrome b5 as a scavenger for the menasemiquinone radical (MD*-) was stoichiometrically reduced by the nNOS reductase domain in the presence of MD, but not of QR. These results indicate that the nNOS reductase domain can catalyze a only one-electron reduction of bivalent quinones. In the presence or absence of Ca2+/CaM, the semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone did not react rapidly with MD, but the fully reduced species of both flavins, FAD and FMN, could donate one electron to MD. The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsuda, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data suggest that the enzyme functions between the 1e- <==> 3e- level during one-electron reduction of MD, and that the rates of quinone reductions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM.     

Detection of S-nitrosothiols

Background

S-nitrosothiols have been recognized as biologically-relevant products of nitric oxide that are involved in many of the diverse activities of this free radical.

Scope of review

This review serves to discuss current methods for the detection and analysis of protein S-nitrosothiols. The major methods of S-nitrosothiol detection include chemiluminescence-based methods and switch-based methods, each of which comes in various flavors with advantages and caveats.

Major conclusions

The detection of S-nitrosothiols is challenging and prone to many artifacts. Accurate measurements require an understanding of the underlying chemistry of the methods involved and the use of appropriate controls.

General significance

Nothing is more important to a field of research than robust methodology that is generally trusted. The field of S-nitrosation has developed such methods but, as S-nitrosothiols are easy to introduce as artifacts, it is vital that current users learn from the lessons of the past. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis

The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.     

One-electron reduction of an anthracycline antibiotic carminomycin by a human erythrocyte redox chain

Human erythrocyte membranes catalyse the NAD(P)H-dependent generation of the semiquinone of an adriamycin-type antibiotic carminomycin under anaerobic conditions. The maximal yield of the antibiotic radical is about 4-fold higher in the presence of NADPH than of NADH. The possible significance of the antibiotic reduction to the semiquinone by a human erythrocyte membrane redox chain for the clinical usage of these antibiotics is discussed.     

Enzymatic reduction of synthetic hemin to heme and its application to study on oxygenation in aqueous medium

The enzyme system consisting of glucose-6-phosphate, glucose-6-phosphate dehydrogenase, ferredoxin, ferredoxin-NADP-reductase, and NADP was used to reduce various synthetic iron(III) porphyrins to iron(II) in aqueous buffer (pH7.0) at 25°C. The oxygenation reactions of the thus prepared iron(II) porphyrin complexes were examined and it was found that only the iron(II) picket fence porphyrin-mono(1-lauryl-2-methylimidazole) complex incorporated in liposomes of phosphatidylcholine can form a stable oxygen adduct at 25°C in neutral aqueous medium.     

One-electron reduction of flavoproteins: pulse radiolysis of chicken egg white riboflavin binding protein

Reduction of the chicken egg white riboflavin binding protein by the hydrated electron results in competitive formation of both a disulfide-electron adduct and an anionic flavin semiquinone bound to the protein. The former decays to products that cannot be observed under the conditions of our experiments. The latter is rapidly protonated to the stable neutral semiquinone. The pH dependence of the rate constant associated with this protonation suggests that an acid/base group on the protein donates a proton to the anionic semiquinone.     

Metalloprotein-dependent decomposition of S-nitrosothiols: studies on the stabilization and measurement of S-nitrosothiols in tissues

The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (L-CysNO, D-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of L-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80 degrees C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of L-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of L-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.     

Enzymatic reduction of synthetic polymer-bound hemin derivatives: efficient method to prepare a heme-oxygen adduct in cooled aqueous medium

Synthetic polymer-bound hemin (iron(III) protoporphyrin IX) derivatives were effectively reduced by ferredoxin and ferredoxin-NADP reductase system. The resultant polymer-bound heme (iron(II) protoporphyrin IX) derivatives formed oxygen adducts with a lifetime of ca. 1 hr in aqueous solution at -30 degrees C. The reduction rate is discussed in terms of the structure of the hemin derivatives.