The acceptor specificity of flavins and flavoproteins. I. Techniques for anaerobic spectrophotometry

1. Easily constructed apparatus is described for spectrophotometry under strictly anaerobic conditions without requiring special cuvettes. It permits the addition of several reagents successively without opening the system to the air.

2. The absorption spectrum of dithionite shows a strong peak at 314 nm, the molar absorbance of which has been determined. This gives a convenient method for the titration of acceptors with dithionite.

3. One molecule of dithionite reacts very rapidly with one molecule of O₂ in solution. The O₂ is reduced quantitatively to H₂O₂. With excess of dithionite another, much slower, reaction follows, in which a second molecule of dithionite is oxidized by the peroxide.

4. A study has been made of the reduction by dithionite of a variety of acceptors commonly used in the study of flavoproteins. The majority react very rapidly, but a few are reduced relatively slowly or not at all.

5. The majority of acceptors do not react significantly with sulphite, the oxidation product of dithionite. One molecule of dithionite then provides two reduction equivalents. A few acceptors, however, react with the sulphite formed, giving a second reaction involving two more equivalents.     

The acceptor specificity of flavins and flavoproteins. III. Flavoproteins

1. The specificity of flavoproteins towards acceptors has been rather neglected, but an attempt is here made to construct a comparative table of acceptor specificities of those flavoprotein enzymes for which data exist.

2. The acceptor specificity of reduced flavin groups, when combined with apoenzyme proteins, is quite different from that of the same flavin groups in the free state (see Part II). Free flavins react very rapidly with a wide range of acceptors, but the same groups combined as flavoproteins have a severely restricted range of action.

3. There are remarkable differences between different flavoproteins. Nearly every flavoprotein fails altogether to react with at least one, and often several, of the acceptors, giving a specificity pattern which is different in each case. There seems to be no general acceptor for flavoproteins.

4. The effect of combination of a flavin with a particular apoenzyme is to inhibit specifically the reaction of the flavin with particular acceptors with which it would react very rapidly in the absence of the apoenzyme.

5. Each apoenzyme produces its own distinctive pattern of inhibitions. The degree of inhibition is often very high; the table shows over 50 cases of specific inhibitions that are essentially complete. Some of these are very difficult to explain.

6. There is no obvious parallelism between any acceptor and any other in its pattern of reactivity with a series of different flavoproteins.

7. In a few cases combination with apoenzyme specifically accelerates the reaction of the flavin with particular acceptors, so that the flavoprotein is oxidized faster than the free flavin.

8. Possible correlations are discussed between the effects of apoenzymes on the reactivity of flavins with acceptors and a number of special known features of different apoenzymes, but no adequate explanation of the differences in specificity has emerged.

9. In view of the interesting nature of the effects, a plea is made for a more intensive study of the acceptor side of flavoprotein specificity.     

Cytochrome oxidase and its derivatives. VIII. Formation and properties of the ‘oxygenated’ from of cytochrome oxidase and its reconversion to ferrous and ferric oxidase

1. The ‘oxygenated’ compound of cytochrome c oxidase used in our experiments is more stable than the compound of previous reports. It is quantitatively reversible to ferrous oxidase.

2. It is best formed with an excess of O₂ after reduction with a minimum amount of dithionite. It can also be formed at low O₂ tension, but then contains some ferric oxidase.

3. Its formation from ferrocyanide-reduced oxidase remains incomplete and subsequent reduction by dithionite is also incomplete.

4. Cyanide does not inhibit its formation from ferrous oxidase. If only ferricytochrome a but no ferricytochrome a₃ is reduced in the presence of cyanide by dithionite, there is no reaction with O₂.

5. The anaerobic reduction of ‘oxygenated’ oxidase by dithionite is monophasic and fast. In contrast, that of ferric oxidase is biphasic, with an initial fast reduction of ferricytochrome a followed by a much slower reduction of ferricytochrome a₃. The rate of cytochrome a, but not that of cytochrome a₃ reduction depends on dithionite concentration.

6. In the presence of dissolved O₂, the ferric oxidase reduction comes to a temporary standstill when one-third of the absorbance increase at 444 mμ has been reached.

7. Ethyl hydrogen peroxide reacting with ferrous oxidase forms a compound similar to the ‘oxygenated’ compound.

8. Hydrogen donors known to react with peroxidase-H₂O₂ complexes, particularly pyrogallol, accelerate the transformation of ‘oxygenated’ to ferric oxidase, though not at a rate comparable to that of cytochrome c.

9. These results strengthen the evidence for cytochromes a and a₃ but indicate that this difference has disappeared in ‘oxygenated’ oxidase.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Autocatalytic peroxidation of ferrocytochrome c

1. Ferricytochrome c acts as a catalyst in the peroxidation of ferrocytochrome c thereby giving rise to an autocatalytic reaction.

2. The rate of the peroxidation reaction is proportional to the concentration of H₂O₂ and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.

3. Integration of the rate equation, d[c³⁺]/dt = k[c³⁺][H₂O₂], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.

4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H₂O₂ derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.

5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H₂O₂, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form.     

Nitroxide-Stimulated H2O2 Decomposition by Peroxidases and Pseudoperoxidases

Nitroxide free radicals interact with Hb/metHb, Mb/metMb and with peroxidases/phenols to induce a catalase-like conversion of H₂O₂ to O₂ (catalatic activity), without being substantially consumed in the process. The mechanism of this reaction is postulated to involve a one-electron oxidation of the nitroxide to the immonium oxene, which then reacts further to release oxygen and the nitroxide. An involvement of the immonium oxene in the reaction mechanism is consistent with ferryl heme reduction by nitroxides and a detection of the reduced nitroxide when the reaction mixture is supplemented with the two-electron reductant sodium borohydride. The nitroxide-induced catalatic activity is completely inhibited when the reaction mixture is supplemented with glutathione. Nitroxides suppress free radical formation by hydroperoxide-activated heme proteins, as inferred from their inhibition of the spin-trapping of glutathionyl radicals. H₂O₂ decomposition and a suppression of reactive free radical formation by heme proteins appears to be an antioxidant activity of nitroxides, which is distinct from their previously reported superoxide dismutating activity and which may be a factor in their protective action in models of cardiac reperfusion injury.     

A comparative Mössbauer study of the mineral cores of human H-chain ferritin employing dioxygen and hydrogen peroxide as iron oxidants

Ferritins are ubiquitous iron storage and detoxification proteins distributed throughout the plant and animal kingdoms. Mammalian ferritins oxidize and accumulate iron as a ferrihydrite mineral within a shell-like protein cavity. Iron deposition utilizes both O₂ and H₂O₂ as oxidants for Fe²⁺ where oxidation can occur either at protein ferroxidase centers or directly on the surface of the growing mineral core. The present study was undertaken to determine whether the nature of the mineral core formed depends on the protein ferroxidase center versus mineral surface mechanism and on H₂O₂ versus O₂ as the oxidant. The data reveal that similar cores are produced in all instances, suggesting that the structure of the core is thermodynamically, not kinetically controlled. Cores averaging 500 Fe/protein shell and diameter  2.6 nm were prepared and exhibited superparamagnetic blocking temperatures of 19 and 22 K for the H₂O₂ and O₂ oxidized samples, respectively. The observed blocking temperatures are consistent with the unexpectedly large effective anisotropy constant K_eff = 312 kJ/m³ recently reported for ferrihydrite nanoparticles formed in reverse micelles [E.L. Duarte, R. Itri, E. Lima Jr., M.S. Batista, T.S. Berquó and G.F. Goya, Large Magnetic Anisotropy in ferrihydrite nanoparticles synthesized from reverse micelles, Nanotechnology 17 (2006) 5549–5555.]. All ferritin samples exhibited two magnetic phases present in nearly equal amounts and ascribed to iron spins at the surface and in the interior of the nanoparticle. At 4.2 K, the surface spins exhibit hyperfine fields, H_hf, of 436 and 445 kOe for the H₂O₂ and O₂ samples, respectively. As expected, the spins in the interior of the core exhibit larger H_hf values, i.e. 478 and 486 kOe for the H₂O₂ and O₂ samples, respectively. The slightly smaller hyperfine field distribution DH_hf for both surface (78 kOe vs. 92 kOe) and interior spins (45 kOe vs. 54 kOe) of the O₂ sample compared to the H₂O₂ samples implies that the former is somewhat more crystalline.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

Particulate formate oxidase from Nitrobacter agilis

1. The nitrite oxidase particles obtained by sonic oscillation of Nitrobacter agilis cells also possessed appreciable formate oxidase activity, ranging from about 25 to 50% of the nitrite oxidase activity depending upon the N. agilis strain. Both activities distributed themselves in the same pattern and proportions during differential centrifugation, and resided solely in the pellet resulting from high-speed centrifugation.

2. Difference spectra of formate-reduced particles or intact cells demonstrated the presence of cytochromes of the c- and a-types like those of the NO₂⁻-reduced material. Under anaerobic conditions NO₃⁻ or fumarate acted as an alternate electron acceptor in place of O₂ in formate oxidation. Under aerobic conditions increasing NO₃⁻ concentrations resulted in (a) an increased role of NO₃⁻ as a terminal electron acceptor compared to O₂, (b) a greater total enzymatic transfer of electrons from formate than if O₂ were the sole electron acceptor, and (c) a partial inhibition of O₂ uptake suggestive of a competition for electrons by the two acceptors. The formate oxidase system failed to catalyze consistently the transfer of electrons to either added mammalian cytochrome c or Fe(CN)₆³⁻. The marked sensitivity of the system to certain inhibitors implicated cytochrome oxidase as an integral part of the formate oxidase. The system was also inhibited significantly by a variety of chelating agents, indicating a metal component in the formate dehydrogenase or early portion of the electron transfer sequence.

3. The stoichiometry of the formate oxidase system was shown to approach the theoretical value of 2 moles of CO₂ evolved per mole of O₂ or per 2 moles of formate consumed.

4. To a limited extent, phosphorylation occurred concomittantly with the oxidation of formate in the presence of the cell-free particulate system.     

Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O₂-) and its dismutation product, hydrogen peroxide (H₂O₂). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 μg/ml) decreased H₂O₂ yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H₂O₂ release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O_2;^- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H₂O₂ and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O₂^- and H₂O₂ generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O_2;^-and *NO production.     

Formation of Hydroxyl Radicals in Biological Systems. Does Myoglobin Stimulate Hydroxyl Radical Formation from Hydrogen Peroxide?

Incubation of horse-heart oxymyoglobin or metmyoglobin with excess H₂O₂ causes formation of myoglobin(IV), followed by haem degradation. At the time when haem degradation is observed, hydroxyl radicals (.OH) can be detected in the reaction mixture by their ability to degrade the sugar deoxyribose. Detection of hydroxyl radicals can be decreased by transferrin or by OH scavengers (mannitol, arginine, phenylalanine) but not by urea. Neither transferrin nor any of these scavengers inhibit the haem degradation. It is concluded that intact oxymyoglobin or metmyoglobin molecules do not react with H₂O₂ to form OH detectable by deoxyribose, but that H₂O₂ eventually leads to release of iron ions from the proteins. These released iron ions can react to form OH outside the protein or close to its surface. Salicylate and the iron chelator desferrioxamine stabilize myoglobin and prevent haem degradation. The biological importance of OH generated using iron ions released from myoglobin by H₂O₂ is discussed in relation to myocardial reoxygenation injury.     

A light-dependent oxygen-reducing system from Anabaena variabilis

Photosynthetic lamellae from Anabaena variabilis catalyze a vigorous photoreduction of O₂ to H₂O₂. The electrons come to O₂ from an artificial donor, reduced 2,3′,6-trichlorophenolindophenol, and the reaction does not require an exogenous autoxidizable substance. Evidence is presented to show that reduced 2,3′,6-trichlorophenolindophenol can donate electrons at two distinct sites. Photoreduction of O₂ is inhibited by antibodies which block the function of the ferredoxin-reducing substance. The O₂-reducing system may result from the formation of a reduced photoproduct which is more accessible to autoxidation than is the analogous product formed in higher plant chloroplasts.     

Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress?

Trehalose is known to protect membranes and macromolecules. Its accumulation has been implicated in allowing plants to tolerate stress, including heat-shock. However, under heat-shock, it is not clear whether trehalose eliminates reactive oxygen species (ROS) directly or indirectly by protecting antioxidant enzymes. In this study, we initially examined the effects of trehalose on the activities of key antioxidant enzymes, including superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate peroxidases (APX) from wheat (Triticum aestivum L.), and then measured the ability of trehalose to scavenge hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻). Our results indicated that trehalose protected SOD activity slightly. However, it inhibited CAT and APX activities under heat stress, with a little protection of CAT activity (only about 7% promotion) at 22 °C. Moreover, trehalose scavenged H₂O₂ and O₂⁻ greatly in a concentration-dependent manner, reaching the maximal scavenging H₂O₂ rate of 95% and O₂⁻ rate of 78%, respectively, at 50 mM trehalose. These results suggest that trehalose plays a direct role in eliminating H₂O₂ and O₂⁻ in wheat under heat stress.     

Inactivation of alcohol dehydrogenase by piroxicam-derived radicals

Alcohol dehydrogenase (ADH) was used as a marker molecule to clarify the mechanism of gastric mucosal damage as a side effect of using piroxicam. Piroxicam inactivated ADH during interaction of ADH with horseradish peroxidase and H₂O₂ (HRP-H₂O₂). The ADH was more easily inactivated under aerobic than anaerobic conditions, indicating participation by oxygen. Superoxide dismutase, but not hydroxyl radical scavengers, inhibited inactivation of ADH, indicating participation by superoxide. Sulfhydryl (SH) groups in ADH were lost during incubation of piroxicam with HRP-H₂O₂. Adding reduced glutathione (GSH) efficiently blocked ADH inactivation. Other SH enzymes, including creatine kinase and glyceraldehyde-3-phosphate dehydrogenase, were also inactivated by piroxicam with HRP-H₂O₂. Thus SH groups in the enzymes seem vulnerable to piroxicam activated by HRP-H₂O₂. Spectral change in piroxicam was caused by HRP-H₂O₂. ESR signals of glutathionyl radicals occurred during incubation of piroxicam with HRP-H₂O₂ in the presence of GSH. Under anaerobic conditions, glutathionyl radical formation increased. Thus piroxicam free radicals interact with GSH to produce glutathionyl radicals. Piroxicam peroxyl radicals or superoxide, or both, seem to inactivate ADH. Superoxide may be produced through interaction of peroxyl radicals with H₂O₂. Thus superoxide dismutase may inhibit inactivation of ADH through reducing piroxicam peroxyl radicals or blocking interaction of SH groups with O₂^-, or both. Other oxicam derivatives, including isoxicam, tenoxicam and meloxicam, induced ADH inactivation in the presence of HRP-H₂O₂.