Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

A reaction between the superoxide free radical and lipid hydroperoxide in sodium linoleate micelles

The oxidation of unsaturated fatty acid micelles by the superoxide free radical ($\text{O}{}^{\mathrm{?}}{}_2$), during γ irradiation in the presence of formate, is kinetically distinct from oxidation by hydroxyl free radicals (HO.). The evidence suggests that a direct reaction between ($\text{O}{}^{\mathrm{?}}{}_2$) and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with ($\text{O}{}^{\mathrm{?}}{}_2$), protects the micelles from oxidation, active superoxide dismutase is no more effective than its apoprotein, due to lack of penetration of the micellar environment. We discuss these findings in the light of recent literature, and with reference to their possible significance for biological systems.     

Reactions of oxygen radical species with methional: a pulse radiolysis study.

The reaction of hydroxyl radicals (^?OH) and superoxide anions ($\text{O}{}_2{}^{\mathrm{?}}$) with methional were investigated by pulse-radiolytic methods. The second-order rate constant for the attack of OH was determined at 8.2×10⁹ M^?1 sec^?1. In the case of $\text{O}{}_2{}^{\mathrm{?}}$ a slow first-order decay rate of 5.2×10³ sec^?1 suggests a far less efficient reaction. The transient species were identified by comparison with published results of pulse radiolysis and EPR spectroscopy of model compounds. The mechanism for the oxidation of methional by OH was found to be more complex than a simple fragmentation reaction.     

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H⁺ and O₂ consumption and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and H₂O₂ production. $\text{ΔO}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{:ΔH}{}_2\text{O}{}_2$ ratio is approximately I indicating that about half H₂O₂ results from $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ dismutation, the second half being formed directly. ΔNADPH:ΔH₂O₂ and ΔO₂:ΔH₂O₂ ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH₂O₂):(ΔO₂-ΔH₂O₂) is 2 allows one to consider direct 4-electron O₂ reduction as the major way of water formation rather than endogenous substrate hydroxylation.     

H/2H isotope effect in redox reactions of cytochrome c

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O₂^? and CO₂^? was determined in H₂O and in ²H₂O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H₂O and ²H₂O. An isotope effect with , depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

The requirement for ferric in the initiation of lipid peroxidation by chelated ferrous iron

When certain ferrous chelates are added to lipid, peroxidation of the lipid occurs following a short lag. This suggests that a product of ferrous autoxidation is required to initiate lipid peroxidation. This autoxidation product is apparently ferric iron, rather than the oxygen radicals which also result from ferrous autoxidation. Studies with oxy-radical scavengers and catalase suggest that $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ H₂O₂, or the ·OH are not involved in the initiation reactions, therefore, we propose that a ferrous-dioxygen-ferric chelate complex may be the initiating species.     

Chartreusin,an antitumor glycoside antibiotic,induces DNA strand scission

The interaction of chartreusin with covalently closed circular PM2 phage DNA was studied. The antibiotic caused a single strand scission in the presence of reducing agents, such as dithiothreitol, ascorbic acid or NaBH₄. The degree of DNA breakage was dependent upon the drug concentration. The DNA-cleaving activity was enhanced by ferrous ion; but was completely blocked by catalase and partially by superoxide dismutase. The results suggest that reduction, chelate formation and auto-oxidation of the antibiotic, presumably the 5,12-dione moiety, produce free radicals, including $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and ^?OH, which are capable of inducing DNA strand scission.     

Mechanism of superoxide anion scavenging reaction by bis-(salicylato)-copper (II) complex.

The mechanism of the reaction of bis-(salicylato)-copper(II) with superoxide anion has been studied by utilizing electron paramagnetic resonance and polarographic techniques. The proposed reaction sequence is as follows:

$\text{Cu}\text{(II) +}\text{O}{}_2{}^{\mathrm{?}}\text{←}\text{Cu}\text{(II)}\text{O}{}_2{}^{\mathrm{?}}\text{←}\text{Cu}\text{(I)}\text{O}{}_2{}^{\mathrm{?}}\text{←}\text{Cu}\text{(I) +}\text{O}{}_2$

Using the xanthine-xanthine oxidase system as a superoxide generator, it was found that the concentration of this copper complex for 50% inhibition of the xanthine-cytochrome c reductase activity was about 1000 times more per mole of copper than that of erythrocyte superoxide dismutase.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Involvement of superoxide in the prolyl and lysyl hydroxylase reactions

Four superoxide dismutase active copper chelates, Cu(acetylsalicylate)₂, Cu(salicylate)₂, Cu(lysine)₂ and Cu(tyrosine)₂, proved to be inhibitors of prolyl and lysyl hydroxylase. The kinetics of the inhibition are consistent with the proposal that these compounds dismutated ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ at the active site of the enzymes. The data strongly suggest that ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ is the active form of O₂ in the prolyl and lysyl hydroxylase reactions.     

Iron(II)-bleomycin. Biochemical and spectral properties in the presence of radical scavengers

Consistent with a recent literature report (Repine, J. E. $\text{et}$$\text{al.}$ (1981) $\text{Proc.}$$\text{Nat.}$$\text{Acad.}$$\text{Sci.}$$\text{USA}$$\text{7}\text{?}$$\text{8}\text{?}$, 1001–1003), the release of [³H]-thymine from PM-2 DNA by Fe(II)-H₂O₂-generated ·OH was suppressed by dimethyl sulfoxide. In contrast, DMSO did not affect [³H]-thymine release mediated by Fe(II)-bleomycin. Under aerobic conditions in the presence of $\text{t}$-butyl phenylnitrone, Fe(II)-BLM produces an epr signal that has been presumed to arise by transfer of ·OH or $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ from the “active complex” of bleomycin to the spin trap. Remarkably, high concentrations (80 mM) of PBN had no effect on the ability of Fe(II)-BLM to solubilize [³H]-thymine, although the ability of authentic ·OH to degrade DNA was completely suppressed under these condition. The suproxide dismutase catalyst tetrakis(4-N-methylpyridyl)porphineiron(III) also failed to suppress BLM-mediated DNA degradation. Moreover, the epr signal observed with 1.6 mM Fe(II)-BLM in the presence of 80 mM PBN was found to be much less intense than that produced by 1.6 mM Fe(II) and 290 mM H₂O₂, but equivalent in intensity to that obtained with 45 mM Fe(II) and exoess H₂O₂. We conclude that the fragmentation of DNA produced by Fe(II)-BLM can be due neither to free ·OH nor to $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. We suggest that DNA degradation is initiated by an “active complex” consisting of BLM, metal and oxygen that functions by abstracting H· from susceptible sites on DNA.     

Significance of O2 availability and cycling on the respiratory burst response of human PMN's exposed to cytochrome c and superoxide dismutase

In many instances the effect of superoxide ($\text{O}{}_2{}^{\mathrm{?}}$) trapping agents in suppressing the net rate of O₂ consumption of activated PMN's is not in accordance with theoretical expectations. We offer here an alternate explanation to those previously presented by Segal and Meshulam (FEBS Letters 100, 27–32) and Babior (Biochem. Biophys. Res. Comm. 91, 222–226). The paradoxical results previously presented can be explained by recognizing that shortly after activation of resting cells an O₂ diffusion layer is established at or near the outer surface of these cells. The presence of this diffusion layer can markedly alter the anticipated stochiometric relationship between $\text{O}{}_2{}^{\mathrm{?}}$ trapped and apparent O₂ consumed by these cells when they are exposed to $\text{O}{}_2{}^{\mathrm{?}}$ trapping agents.     

The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system

The method of determination of Redox potentials of radicals, using the pulse radiolysis technique, is outlined. The method is based on the determination of equilibrium constants of electron transfer reactions between the radicals and appropriate acceptors. The limitations of this technique are discussed.The redox potentials of several quinones-semiquinones are calculated, as well as the standard redox potential of the peroxy radical. and the redox oxidation properties of the peroxy radical in various systems and pH are discussed. The value determined for the redox potentials of $\text{O}{}_2\text{O}{}_2{}^{\mathrm{?}}$ is higher by more than 0.2 V than earlier estimates, which has important implications on the possible role of O₂^? in biological processes of O₂ fixation.     

Photobiochemistry in the dark: photohemolysis of red cells sensitized by chlorpromazine-bioenergized triplet acetone system

Chlorpromazine is decomposed when it is treated with bioenergized triplet acetone from the 2-methylpropanal/red cells/O₂ system, forming chlorpromazine-5-oxide, with a concomitant strong hemolytic effect observed by a spectrophotometric method. Experiments with external superoxide dismutase, catalase, benzoate and bicarbonate indicate the absence of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$, H₂O₂ and OH· species as the precursor of the hemolytic effect.Comparison between the 2-methylpropanal/peroxidase/O₂ system and the 2-methylpropanal/red cells/O₂ system in the presence of chlorpromazine, indicate that essentially the same type of mechanism occurs in both cases.These results could explain the $\text{in}$$\text{vivo}$ hemolytic and toxic effect of chlorpromazine in the dark.     

Evidence for a coordination position available to solute molecules on one of the metals at the active center of reduced bovine superoxide dismutase

We have measured the contribution of the reduced form of bovine $\text{Zn}\text{Cu}$ superoxide dismutase to the relaxation of the ³⁵Cl nucleus of chloride ion. The reduced protein has a molar relaxivity approximately 2.5 greater than the metal free protein, and addition of a small excess of cyanide lowers the relaxivity of the reduced protein to that of the apo-protein. We have interpreted these observations in terms of an open coordination position on one of the two metal ions, and we have proposed a mechanism for the reduction of superoxide by reduced superoxide dismutase which requires that $\text{O}{}_2{}^{\mathrm{?}}$ binds to Cu⁺ prior to electron transfer.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.