Role of extra- and intracellular Ca2+ in changes of NO2- and H2O2 production by endometrium

The work is aimed to find out the role of extra- and intracellular Ca2+ in cyclic mechanisms of NO2- and H2O2 metabolism in the stroma cells of endometrium activated by acetylcholine. High activity of Mg2+, Ca(2+)-ATP-ase is characteristic of stroma cells of the endometrium. This activity is tested in the presence of 0.01% of the Triton X-100 (36 +/- +/- 2 mumole Pi/mg of protein for 1 hour). The acetylcholinesterase activity in these systems is equal to 9.8 +/- 0.2 mumole thiocholinebromide/mg of protein for 1 hour. Acetylcholine (10 microM) elevated essentially the concentration of cytosolic Ca2+ in them. It was established, that in the control the suspension of stroma cells produced 1 nmol/NO2-/10(6) of cells, this value being constant for the experimental period of time in the range of 5-60 s. The activation of cells (1 microM acetylcholine + 10 microM Ca2+) results in the appearance of cyclicity (maxima on 5, 15 and 60 s) and 5-fold intensification of NO2- production. The rise of extracellular concentration of Ca2+ up to 0.1-1--10 mM results in essential change of the character of the time dependence of NO2- metabolism and only in inappreciable intensification of the response amplitude. Such a pattern is observed for H2O2: 0.77 mumol H2O2/10(6) of cells in the control, at 10 microM Ca2+ maxima of production on the 5 and 30 s, change of the form of the time response, instead of the amplitude under the increase of concentration of extracellular Ca2+. Preincubation of cells with modifiers endoplasmic reticulum ryanodine, caffeine (1 mM) and heparine (10 mM) results in essential drop of NO2- production and infringement of cyclicity, the effect of ryanodine being more expressed on 5 s, than on 15 s, and heparine--also on 5 s, and on 15 s. Preincubation of cells with methylene blue (10 mM), which inhibits guanilate cyclase, result in considerable intensification of NO2- and H2O2 formation and cyclicity losses. Dynamics of NO2- formation (is controversy) reciprocated with cGMP, whereas nitrosoglutathione production and NO3- tends to saturation in the course of time. Thus, acetylcholine-dependent variations of NO2- and H2O2 concentrations in the suspension of stroma cells depend on the state of extra- and intracellular Ca(2+)-stores, are controlled by cGMP. It may be assumed, that the NO2- production minima are caused by its transfer in NO3- and its binding with glutathione.     

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.     

Effect of steroid hormones and oxytocin on NO and H2O2 production in the endometrium

The time and concentration regularities of influence of based estrogens (estrone), hystogenes (progesterone) and their analogs (ecdysterone and diethylstilbestrole) on production of NO and H2O2 by endometrium stroma cells. The level of NO2- in the cell suspension was determined on Grees reactive response, and H2O2 by permanganate measurements in paravariation with the use of horse-radisch peroxidase. It is supposed, that the studied steroids in the experiment conditions affect both the constitutive enzyme systems (membrane or nongenomic phase of activity), and the level of a gene expression (actynomycin D sensitive) and their activity is connected with the rising of concentration of cytosolic Ca2+ in cells. It is shown, that progesterone in hormonal concentrations boosts biosynthesis of NO and H2O2, in comparison with estrone, both in real time of signalling events in the cells (5-30 s), and at long-term action (1-3 h) more effectively. The analogs of steroid hormones, ecdysterone and diethylstilbestrole, were considerably by characterized less effect. The obtained results allow to assume a prime role of progesterone in mechanisms of the endometrium-dependent relaxation of the myometrium. Peptide hormone oxytocine reduces NO production by endometrium and destroys the conforming promoting effect of progesterone, that can testify to the important role of endometrium in processes of initiation of contractile activity of a uterus in labors. The form and amplitude of changes of NO and H2O2 concentration specific for each of studied agonists dilate essentially potentialities of the biological information transmission and allow distinguishing more in detail the external signals and to differentiate the response of cells to various external stimulants. Nitrogen oxide plays an essential role in our system both in the real time of signalling events in cells, and under long-term action, whereas hydrogen peroxide--only at the first stage.     

Interrelation between NO and H2O2 formation and their role in the regulation of ion homeostasis of cells

The review deals with some problems of formation, utilization and functioning two of the most important and the lest cytotoxic active oxidative metabolites--NO and H2O2. The interrelation of NO and H2O2 cellular metabolism is reviewed. There are literature data regarding NO and H2O2 significance in the cellular signalling transduction and tissues paracrine regulation. The peculiar attention is dedicated to NO and H2O2 significance in the smooth muscle cells ion homeostasis regulation particularly in the uterus. As well there are the own author's data evidencing about the composite and ambiguous concentration-dependent physiological action of these metabolites. Varying in level in the cells NO and H2O2 may have a cyclic character similar to the oscillations of some other signaling molecules concentrations, evidently playing an important role in the mechanisms of cellular signal transduction and in the paracrine system.     

The regulation of mitochondrial oxygen uptake by redox reactions involving nitric oxide and ubiquinol

The reversible inhibitory effects of nitric oxide (.NO) on mitochondrial cytochrome oxidase and O(2) uptake are dependent on intramitochondrial.NO utilization. This study was aimed at establishing the mitochondrial pathways for.NO utilization that regulate O-(2) generation via reductive and oxidative reactions involving ubiquinol oxidation and peroxynitrite (ONOO(-)) formation. For this purpose, experimental models consisting of intact mitochondria, ubiquinone-depleted/reconstituted submitochondrial particles, and ONOO(-)-supplemented mitochondrial membranes were used. The results obtained from these experimental approaches strongly suggest the occurrence of independent pathways for.NO utilization in mitochondria, which effectively compete with the binding of.NO to cytochrome oxidase, thereby releasing this inhibition and restoring O(2) uptake. The pathways for.NO utilization are discussed in terms of the steady-state levels of.NO and O-(2) and estimated as a function of O(2) tension. These calculations indicate that mitochondrial.NO decays primarily by pathways involving ONOO(-) formation and ubiquinol oxidation and, secondarily, by reversible binding to cytochrome oxidase.     

Nitric oxide and mitochondrial complex IV

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.     

Involvement of the mitochondrial K(+)ATP channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures

Soybean suspension cell cultures were treated by H2O2 or nitric oxide (NO), to assess the mechanism leading to programmed cell death (PCD). Hydrogen peroxide (5 mM) induced PCD. Cells become necrotic at 20 mM H2O2, with cells exhibiting intermediate hallmarks before that (necrapoptotic cells). The level of ATP and of glucose-6-phosphate remained constant in cells undergoing PCD, while it decreased significantly in the necrotic ones. Mitochondria, isolated from 5 mM H2O2-treated (apoptotic) cells, showed that succinate-dependent oxygen consumption was slightly uncoupled, and the electrical potential difference (delta psi) weakly decreased. The addition of KCl to the delta psi formed determined a partial dissipation, which was higher than the dissipation observed in mitochondria from control cells. The addition of cyclosporin A (CsA) to de-energized mitochondria also induced delta psi formation, due to a K+ efflux from the matrix, which was decreased in mitochondria from treated cells. The same pattern of response was also observed in mitochondria isolated from 1 mM sodium nitroprusside (NO)-treated cells, exhibiting apoptotic symptoms. In mitochondria isolated from 20 mM H2O2-treated (necrotic) cells, succinate-dependent oxygen consumption was completely uncoupled, delta psi generation significantly inhibited, and CsA-dependent delta psi formation prevented. In addition, mitochondria isolated from control cells still underwent swelling, which was partially or completely prevented in mitochondria isolated from apoptotic or necrotic cells, respectively. The moderate swelling was accompanied by a slight rupture of the outer membrane and by a release of cytochrome c. These results point to the involvement of a K(+)ATP channel during the manifestation of PCD induced by H2O2 or NO in plants.     

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.     

Co-oxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H2O2

The reaction between metmyoglobin and H2O2 proceeds with oxidation of the hemo-protein iron to a higher valence state and consumption of the peroxide. This reaction is further associated with (a) O2 evolution; (b) hydroxylation of the aromatic compound salicylate to yield a set of dihydroxybenzoic acid derivatives (analyzed by HPLC with electrochemical detection); (c) autoxidation of cholesterol with formation of 3 beta-hydroxy-5-alpha-cholest-6-ene-5-hydroperoxide; and (d) formation of electronically excited states detected by low-level chemiluminescence. The heterolytic scission of the O-O bond of hydroperoxides by metmyoglobin causes the formation of an oxidizing equivalent capable of promoting peroxidation of linoleate and arachidonate (as indicated by the parallel formation of thiobarbituric acid-reactive material and an enhancement of chemiluminescence intensity). The identity of the oxidizing equivalent(s) is discussed in terms of the formation of a relatively stable higher state of oxidation of heme Fe (FeIV-OH or FeV = O) as well as on possible intermediate species derived during the decomposition of H2O2 by metmyoglobin, such as HO.and 1O2. These species might be involved either simultaneously or sequentially in the peroxidation of fatty acids as well as in the tissue damage associated with the formation of H2O2 in ischemic-reperfusion states.     

Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(?-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(?)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(?-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (?)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.     

Salidroside inhibits H2O2-induced apoptosis in PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

We used a rat pheochromocytoma (PC12) cell line to study the effects of salidroside on hydrogen peroxide (H(2)O(2))-induced apoptosis. In PC12 cells, H(2)O(2)-induced apoptosis was accompanied by the down-regulation of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol. Moreover, salidroside attenuated caspase-3, -8 and -9 activation, and eventually protected cells against H(2)O(2)-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with salidroside can block H(2)O(2)-induced apoptosis by regulating Bcl-2 family members and by suppressing cytochrome c release and caspase cascade activation.     

Effect of cytochrome c on the generation and elimination of O2*- and H2O2 in mitochondria

The primary recognized function of cytochrome c is to act as an electron carrier transferring electrons from complex III to complex IV in the respiratory chain of mitochondria. Recent studies on cell apoptosis reveal that cytochrome c is responsible for the programmed cell death when it is released from mitochondria to cytoplasm. In this study we present evidence showing that cytochrome c plays an antioxidative role by acting on the generation and elimination of O(2)(*) and H(2)O(2) in mitochondria. The O(2)(*) and H(2)O(2) generation in cytochrome c-depleted Keilin-Hartree heart muscle preparation (HMP) is 7-8 times higher than that in normal HMP. The reconstitution of cytochrome c to the cytochrome c-depleted HMP causes the O(2)(*) and H(2)O(2) generation to exponentially decrease. An alternative electron-leak pathway of the respiratory chain is suggested to explain how cytochrome c affects on the generation and elimination of O(2)(*) and H(2)O(2) in mitochondria. Enough cytochrome c in the respiratory chain is needed for keeping O(2)(*) and H(2)O(2) at a lower physiological level. A dramatic increase of O(2)(*) and H(2)O(2) generation occurs when cytochrome c is released from the respiratory chain. The burst of O(2)(*) and H(2)O(2), which happens at the same time as cytochrome c release from the respiratory chain, should have some role in the early stage of cell apoptosis.     

Oxidative modification of cytochrome P-450 during its function. I. Comparative study of the inactivation of cytochrome P-450 LM2 in various systems

The changes in the content of purified isolated cytochrome P-450 LM2 under the action of hydrogen peroxide and during its operation in a soluble reconstituted system were studied. It was found that cytochrome P-450 LM2 inactivation by hydrogen peroxide is accompanied by a decrease in the hemoprotein activity, loss of heme, oxidation of SH-groups and changes in the oligomeric state of the enzyme. There were some differences in the mechanisms of cytochrome P-450 LM2 inactivation under the action of H2O2 and during catalysis.     

Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H2O2 by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H2O2 in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2',7'-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2',7'-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H2O2. By examining the effects of radical scavengers on 2',7'-dichlorofluorescin oxidation by cytochrome c/H2O2, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

The antioxidant functions of cytochrome c

Low (C(1/2) = 1.5 x 10(-7) M) concentrations of horse cytochrome c strongly inhibit H(2)O(2) production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD(+). The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD. Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect. H(2)O(2) formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H(2)O(2) production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C(1/2) is about 5 x 10(-7) M). In vesicles, SOD abolishes the cytochrome c inhibition. We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H(2)O(2) production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e. the oxidation of O(2-*) to O(2). A possible role of cytochrome c in the antioxidant defence is discussed.     

Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging

The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.     

Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from cytochrome c-derived tyrosyl radical

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.