Effects of dopamine beta-monooxygenase substrate analogs on ascorbate levels and norepinephrine synthesis in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to investigate the effects of prototypic dopamine beta-monooxygenase substrate analogs of two distinct classes on intravesicular reduced ascorbic acid (AscH2) levels and on norepinephrine synthesis. Phenyl-2-aminoethyl sulfide (PAES), a sulfur-containing substrate, was shown to concentrate within ghosts, a process that was time and ATP dependent, but reserpine insensitive. Dopamine beta-monooxygenase oxygenation of PAES resulted in accumulation of the oxygenation product, PAESO, without affecting intravesicular levels of AscH2. Similarly, incubations of ghosts with phenyl-2-aminoethyl selenide (PAESe) also resulted in rapid, time- and ATP-dependent, but reserpine-insensitive uptake. However, oxygenation of PAESe by dopamine beta-monooxygenase within ghosts was found to cause a marked decrease in intravesicular AscH2, without buildup of the oxygenated product, phenyl 2-aminoethyl selenoxide. These results illustrate two basic differences between the consequences of PAES and PAESe turnover: while PAES accumulation proceeds concomitant with PAESO production and without AscH2 depletion, PAESe accumulation proceeds with a marked lowering of internal AscH2 but without observable product formation. Both PAES and PAESe were capable of competing with dopamine, the physiological substrate, for enzymatic oxygenation and/or vesicular uptake, and were capable of significantly reducing norepinephrine synthesis. In experiments where ghosts were preincubated with either PAES or PAESe with delayed addition of dopamine, it was clear that neither compound nor their oxygenated products interfered with electron transport via cytochrome b561. These results are consistent with the hypothesis that the physiological activity observed with both PAES and PAESe may be related to their ability to gain entrance to adrenergic neurons and decrease norepinephrine synthesis within neurotransmitter storage vesicles.     

Reaction of phenylaminoethyl selenides with peroxynitrite and hydrogen peroxide

Peroxynitrite, a reactive cytotoxic species generated by the reaction of superoxide with nitric oxide, rapidly oxidizes phenylaminoethyl selenide (PAESe) and its para-substituted derivatives with second-order rate constants ranging from 900 to 3000 M(-1) s(-1) at neutral pH (pH 7.0) and 25 degrees C. These values are approximately 3 x 10(4) times greater than the corresponding rate constants for the reactions of selenides with hydrogen peroxide. The peroxynitrite reaction was also studied at alkaline pH. HPLC analysis confirms that both the peroxynitrite and hydrogen peroxide reactions produced the corresponding phenylaminoethyl selenoxide (PAESeO) as the sole selenium-containing product, with a stoichiometry of 1 mol of PAESe oxidized per 1 mol of PAESeO formed per 1 mol of oxidant reacted. The influence of para-substituents on the rate constants was investigated using Hammett plots; in both cases the data are consistent with an S(N)2-type mechanism, wherein the selenium atom acts as the nucleophile. Our results provide further evidence that organoselenium compounds may play a protective role in the defense against the many reactive oxidizing species produced in cellular metabolism.     

The antioxidant phenylaminoethyl selenide reduces doxorubicin-induced cardiotoxicity in a xenograft model of human prostate cancer

Anthracyclines are potent anticancer agents, but cardiotoxicity mediated by free radical generation limits their clinical use. This study evaluated the anticancer activity of phenyl-2-aminoethyl selenide (PAESe) and its potential to reduce doxorubicin (DOX)-induced cardiotoxicity. Growth inhibitory effects of PAESe with DOX, and vincristine, clinically used anticancer agents, and tert-butylhydroperoxide (TBHP), a known oxidant, on the growth of human prostate carcinoma (PC-3) cells was determined. PAESe (≤1μm) did not alter the growth of PC-3 cells, however, concomitant use of PAESe decreased the oxidative-mediated cytotoxicity of TBHP, but had limited effect on vincristine or DOX activity. Further, PAESe decreased the formation of intracellular reactive oxygen species from TBHP and DOX. The effect of PAESe on the activity of DOX was determined using a tumor (PC-3) xenograft model in mice. PAESe did not alter DOX antitumor activity and showed evidence of direct antitumor activity relative to controls. DOX treatment decreased mice body weight significantly, whereas concomitant administration of PAESe and DOX was similar to controls. Most importantly, PAESe decreased DOX-mediated infiltration of neutrophil and macrophages into the myocardium. These data suggest PAESe had in vivo antitumor activity and in combination with DOX decreased early signs of cardiotoxicity while preserving its antitumor activity.     

Olefin oxygenation and N-dealkylation by dopamine beta-monooxygenase: catalysis and mechanism-based inhibition

In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.     

Cytochrome b₅ reductase-cytochrome b₅ as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b? reductase-cyt b?. In fungi, limited information is available for the cyt b(5) reductase-cyt b(5) system. Here we characterized the kinetic mechanism of (cyt b?r)-cyt b? redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b?r followed a "ping-pong" mechanism and could directly reduce cytochrome c. However, unlike other cyt b? reductases, Pc-cyt b?r lacked the typical ferricyanide reduction activity, a standard for cyt b? reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b?r-cyt b? complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b?r-cyt b? complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b?r-cyt b?) in terms of supporting a P450 monooxygenase activity.     

Discrimination of ascorbate-dependent nonenzymatic and enzymatic,membrane-bound reduction of nitric oxide in denitrifying Pseudomonas perfectomarinus

The marine nitrite-respiring (denitrifying) bacterium, Pseudomonas perfectomarinus, catalyzes by a membrane-bound enzyme the reduction of nitric oxide to nitrous oxide with ascorbate-reduced phenazine methosulfate as electron donor. The entire nitric oxide-reducing capability of a cell-free system was membrane bound and this process was studied with respect to pH and substrate dependency. The enzymatic process was perturbed by an identical nonenzymatic reduction by iron(II) ascorbate in neutral to alkaline aqueous solution. 2 mol nitric oxide and 1 mol ascorbate were consumed per mol nitrous oxide formed. Enzymatic and nonenzymatic processes were discriminated by their differential behavior towards pH and metal-chelating agents. The pH optimum for the enzymatic and nonenzymatic reaction was 5.2 and greater than 7.0, respectively. EDTA (10 mM) inhibited the nonenzymatic reduction completely without interfering with the membrane-bound activity. The nonenzymatic system mimics the reaction of nitric oxide reductase and could serve as a model to study the formation of the N-N bond in denitrification. Enzymatic generation of nitric oxide by cytochrome cd and subsequent nonenzymatic reduction to nitrous oxide simulate an overall quasi-enzymatic nitrous oxide formation by cytochrome cd. The nonenzymatic reduction of nitric oxide might have occurred in previous work due to the ubiquitous use of ascorbate in studies on nitrite respiration and the likelihood of adventitious iron in biological samples.     

Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity

Upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL may act as a switch to regulate cyt c's mitochondrial functions. Using cyclic voltammetry and equilibrium redox titrations, we show that the redox potential of cyt c shifts negatively by 350-400 mV upon binding to CL-containing membranes. Consequently, functions of cyt c as an electron transporter and cyt c reduction by Complex III are strongly inhibited. Further, CL/cyt c complexes are not effective in scavenging superoxide anions and are not effectively reduced by ascorbate. Thus, both redox properties and functions of cyt c change upon interaction with CL in the mitochondrial membrane, diminishing cyt c's electron donor/acceptor role and stimulating its peroxidase activity.     

An investigation of the biotransformation of organic selenides by fungi

A series of alkyl phenyl selenides has been incubated with the fungi Aspergillus niger, Aspergillus foetidus, Mortierella isabellina, and Helminthosporium sp. These fungi oxidize the corresponding sulfides to sulfoxides efficiently, but in no case was any evidence obtained that the microbial oxidation of selenide to selenoxide was occurring. The fate of methyl phenyl selenide following incubation with M. isabellina was investigated using methyl-¹⁴C-labeled substrate, and by quantitative selenium analysis. These techniques indicate that the selenide is taken into the fungal cell efficiently and that some metabolic cleavage of the selenium-methyl carbon bond may occur.     

Photoinduced electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the Rhodobacter sphaeroides cytochrome bc(1) complex. Effects of pH,temperature, and driving force

Electron transfer from the Rieske iron-sulfur protein to cytochrome c(1) (cyt c(1)) in the Rhodobacter sphaeroides cytochrome bc(1) complex was studied using a ruthenium dimer complex, Ru(2)D. Laser flash photolysis of a solution containing reduced cyt bc(1), Ru(2)D, and a sacrificial electron acceptor results in oxidation of cyt c(1) within 1 micros, followed by electron transfer from the iron-sulfur center (2Fe-2S) to cyt c(1) with a rate constant of 80,000 s(-1). Experiments were carried out to evaluate whether the reaction was rate-limited by true electron transfer, proton gating, or conformational gating. The temperature dependence of the reaction yielded an enthalpy of activation of +17.6 kJ/mol, which is consistent with either rate-limiting conformational gating or electron transfer. The rate constant was nearly independent of pH over the range pH 7 to 9.5 where the redox potential of 2Fe-2S decreases significantly due to deprotonation of His-161. The rate constant was also not greatly affected by the Rieske iron-sulfur protein mutations Y156W, S154A, or S154A/Y156F, which decrease the redox potential of 2Fe-2S by 62, 109, and 159 mV, respectively. It is concluded that the electron transfer reaction from 2Fe-2S to cyt c(1) is controlled by conformational gating.     

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum.

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.     

Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione‐S‐transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione‐S‐transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.     

Enzymatic methylation of sulfide, selenide, and organic thiols by Tetrahymena thermophila

Cell extracts from the ciliate Tetrahymena thermophila catalyzed the S-adenosylmethionine-dependent methylation of sulfide. The product of the reaction, methanethiol, was detected by a radiometric assay and by a gas-chromatographic assay coupled to a sulfur-selective chemiluminescence detector. Extracts also catalyzed the methylation of selenide, and the product was shown by gas chromatography-mass spectrometry to be methaneselenol. The sulfide and selenide methyltransferase activities copurified with the aromatic thiol methyltransferase previously characterized from this organism (A.-M. Drotar and R. Fall, Pestic. Biochem. Physiol. 25:396-406, 1986), but heat inactivation experiments suggested the involvement of distinct sulfide and selenide methyltransferases. Short-term toxicity tests were carried out for sulfide, selenide, and their methylated derivatives; the monomethylated forms were somewhat more toxic than the nonmethylated or dimethylated compounds. Cell suspensions of T. thermophila exposed to sulfide, methanethiol, or their selenium analogs emitted methylated derivatives into the headspace. These results suggest that this freshwater protozoan is capable of the stepwise methylation of sulfide and selenide, leading to the release of volatile methylated sulfur or selenium gases.     

Enzymatic methylation of sulfide, selenide, and organic thiols by Tetrahymena thermophila.

Cell extracts from the ciliate Tetrahymena thermophila catalyzed the S-adenosylmethionine-dependent methylation of sulfide. The product of the reaction, methanethiol, was detected by a radiometric assay and by a gas-chromatographic assay coupled to a sulfur-selective chemiluminescence detector. Extracts also catalyzed the methylation of selenide, and the product was shown by gas chromatography-mass spectrometry to be methaneselenol. The sulfide and selenide methyltransferase activities copurified with the aromatic thiol methyltransferase previously characterized from this organism (A.-M. Drotar and R. Fall, Pestic. Biochem. Physiol. 25:396-406, 1986), but heat inactivation experiments suggested the involvement of distinct sulfide and selenide methyltransferases. Short-term toxicity tests were carried out for sulfide, selenide, and their methylated derivatives; the monomethylated forms were somewhat more toxic than the nonmethylated or dimethylated compounds. Cell suspensions of T. thermophila exposed to sulfide, methanethiol, or their selenium analogs emitted methylated derivatives into the headspace. These results suggest that this freshwater protozoan is capable of the stepwise methylation of sulfide and selenide, leading to the release of volatile methylated sulfur or selenium gases.     

RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H(2)O(2) or by the Fe(II)/ascorbate/H(2)O(2) system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H(2)O(2) relative to the Fe(II)/ascorbate/H(2)O(2) system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H(2)O(2) concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H(2)O(2), it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.     

Soluble variants of Rhodobacter capsulatus membrane-anchored cytochrome cy are efficient photosynthetic electron carriers

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.     

Photosynthetic cytochrome c550

Cytochrome c550 (cyt c550) is a membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, such as red and brown algae. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl(-) and Ca(2+) ions to the oxygen evolving complex and protects the Mn(4)Ca cluster from attack by bulk reductants. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (E(m)) of the purified soluble form (from -250 to -314mV) is incompatible with a redox function in PSII. However, more positive values for the Em have been obtained for the cyt c550 bound to the PSII. A very recent work has shown an E(m) value of +200mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22?) between cyt c550 and the nearest redox cofactor (Mn(4)Ca cluster), an electron transfer reaction between these components is possible. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed. The aim of this article is to review previous studies done on cyt c550 and to consider its function in the light of the new results obtained in recent years. The emphasis is on the physical properties of the heme and its redox properties. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Fabrication of cytochrome c-poly(5-amino-2-napthalenesulfonic acid) electrode by one step procedure and direct electrochemistry of cytochrome c

Herein, we reported for the first time one step procedure for the preparation of cytochrome c (cyt c)-poly (5-amino-2-napthalenesulfonic acid) (PANS) modified glassy carbon electrode by cyclic voltammetrically (CV). Hereafter, we called the above modified electrode as cyt c-PANS electrode. The presence of cyt c on modified electrode was investigated with electrochemical quartz crystal microbalance (EQCM), CV, and superoxide radicals reaction studies. The reaction between cyt c in the modified electrode and superoxide radicals in solution, was exemplified by cyclic voltammetric measurements. Surface morphology of the modified electrode was investigated by using atomic force microscopy (AFM). The modified electrode showed a pair of well defined redox peak in PBS solution, pH 6.7. The modified electrode utilized for electrocatalytic reduction as well as amperometric determination of hydrogen peroxide (H(2)O(2)). The detection limit and linear range for H(2)O(2) were 5 and 50 microM to 7 mM, respectively.     

Biochemical properties of cytochrome c nitrated by peroxynitrite

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.     

Identification of a thioselenurane intermediate in the reaction between phenylaminoalkyl selenoxides and glutathione

Selenium has a long history of association with human health and disease, and a low concentration of selenium in plasma has been identified in epidemiological studies as a risk factor for several disorders associated with oxidative stress. This association suggests that organoselenium compounds capable of propagating a selenium redox cycle might supplement natural cellular defenses against oxidants, such as peroxynitrite and hydrogen peroxide. While several such organoselenium compounds are under active investigation as potential therapeutic agents, chemical characterization of reaction intermediates involved in their redox cycling has been problematical. We now report evidence that the reaction between phenylaminoalkyl selenoxides and glutathione (GSH) proceeds through the intermediacy of a thioselenurane species. The results of stopped-flow kinetic experiments were consistent with a rapid and stoichiometric initial reaction of GSH with selenoxide to generate a kinetically-detectable intermediate, followed by a slower reaction of this intermediate with a second molecule of GSH to produce the final selenide and GSSG products. Flow injection ESI-MS and ESI-MS/MS experiments confirmed that the reaction intermediate is indeed a thioselenurane. Final structural characterization of the thioselenurane intermediate was obtained from analysis of the daughter ions produced in flow injection ESI-MS/MS experiments. These results help to elucidate the chemical nature of the redox cycling of phenylaminoalkyl selenides, and represent, to our knowledge, the first evidence for the intermediacy of a thioselenurane species in the reaction of thiols with selenoxides.