Oxidation of anthracycline anticancer agents by the peroxidase mimic microperoxidase 11 and hydrogen peroxide

The interaction of two clinically important anticancer agents doxorubicin (DXR) and daunorubicin (DNR) and the DNR analog 5-iminodaunorubicin (5IDNR) with the model mammalian peroxidase microperoxidase 11 (MP11) and H(2)O(2) has been investigated using spectrophotometric and EPR techniques. We demonstrate that DNR, DXR, and 5IDNR undergo irreversible oxidation by MP11/H(2)O(2), forming colorless products in both phosphate buffer pH 7.0 and in phosphate buffer pH 7.0/MeOH mixture (1:1 vol/vol), suggesting an extensive modification of the compounds' chromophores. The initial rate of the anthracyclines' oxidation is independent of anthracycline concentrations, but is linearly dependent on [H(2)O(2)](i) at constant [MP11](i) (and vice versa), indicating that the reaction is zero order in [anthracycline], first order with respect to [H(2)O(2)] and [MP11], and second order overall. Based on data obtained using DNR, DXR, 5IDNR, and p-hydroquinone k(2app), the apparent second order rate constant for the formation of a reactive intermediate from MP11 and H(2)O(2) (an analog of peroxidase compound I) has been determined to be in the range of (2.51-5.11) x 10(3) M(-1) s(-1) in both solvent systems. EPR studies show that oxidation of DNR, DXR, or 5IDNR with MP11/H(2)O(2) generates free radicals, suggesting that the reaction may be a one-electron process. This study also shows that 5IDNR, but not DNR or DXR, efficiently protects MP11 heme against degradation by H(2)O(2). Our overall conclusion is that MP11 is an effective catalyst of oxidation of anthracyclines by H(2)O(2). Given that, at sites of inflammation or cancer, the anthracyclines can colocalize with peroxidases, protein degradation products, and with H(2)O(2), peroxidation could be one possible fate of these anticancer agents in vivo.     

Oxidation of biological electron donors and antioxidants by a reactive lactoperoxidase metabolite from nitrite (NO2-): an EPR and spin trapping study

We report that a lactoperoxidase (LPO) metabolite derived from nitrite (NO2-) catalyses one-electron oxidation of biological electron donors and antioxidants such as NADH, NADPH, cysteine, glutathione, ascorbate, and Trolox C. The radical products of the reaction have been detected and identified using either direct EPR or EPR combined with spin trapping. While LPO/H2O2 alone generated only minute amounts of radicals from these compounds, the yield of radicals increased sharply when nitrite was also present. In aerated buffer (pH 7) the nitrite-dependent oxidation of NAD(P)H by LPO/H2O2 produced superoxide radical, O2*-, which was detected as a DMPO/*O2H adduct. We propose that in the LPO/H2O2/NO2-/biological electron donor systems the nitrite functions as a catalyst because of its preferential oxidation by LPO to a strongly oxidizing metabolite, most likely a nitrogen dioxide radical *NO2, which then reacts with the biological substrates more efficiently than does LPO/H2O2 alone. Because both nitrite and peroxidase enzymes are ubiquitous our observations point at a possible mechanism through which nitrite might exert its biological and cytotoxic action in vivo, and identify some of the physiological targets which might be affected by the peroxidase/H2O2/nitrite systems.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.     

Effects of peroxidase substrates on the Amplex red/peroxidase assay: antioxidant properties of anthracyclines

Oxidation of Amplex red (AR) by H(2)O(2) in the presence of horseradish peroxidase (HRP) gives rise to an intensely colored product, resorufin. This reaction has been frequently employed for measurements of low concentrations of H(2)O(2) in biological samples. In the current study, we show that alternative peroxidase substrates, such as p-hydroquinone, acetaminophen, anticancer mitoxantrone, and ametantrone, inhibit AR oxidation by consuming H(2)O(2) in a competitive process. In contrast, the anthracycline agents doxorubicin, daunorubicin, and 5-iminodaunorubicin are markedly less efficient as competitors in these reactions, as is salicylic acid. When [H(2)O(2)]>[AR], the generated resorufin was oxidized by HRP and H(2)O(2). In the presence of anthracyclines, this process was inhibited and occurred with a lag time, the duration of which depended on the concentration of anthracycline. We propose that the mechanism of this inhibition is due to the antioxidant activity of anthracyclines involving the reduction of the resorufin-derived phenoxyl radical by the drugs' hydroquinone moiety back to resorufin. In addition to HRP, lactoperoxidase, myeloperoxidase, and HL-60 cells, naturally rich in myeloperoxidase, also supported these reactions. Results of this study suggest that extra caution is needed when using AR to measure cellular H(2)O(2) in the presence of alternative peroxidase substrates. They also demonstrate that the anticancer anthracyclines may function as antioxidants.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

Lactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide: 15N nuclear magnetic resonance and optical spectral studies

To establish the agent(s) responsible for the activity of the lactoperoxidase (LPO)/SCN-/H2O2 system, the oxidation of thiocyanate with hydrogen peroxide, catalyzed by lactoperoxidase, has been studied by 15N NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pHs. The formation of hypothiocyanite ion (OSCN-) as one of the oxidation products correlated well with the activity of the LPO/SCN-/H2O2 system and was maximum when the concentrations of the H2O2 and SCN- were nearly the same and the pH was less than 6.0. At [H2O2]/[SCN-] = 1, OSCN- decomposed very slowly back to thiocyanate. When the ratio [H2O2]/[SCN-] was above 2, formation of CN- was observed, which was confirmed by 15N NMR and also by changes in the optical spectrum of LPO. The oxidation of thiocyanate by H2O2 in the presence of LPO does not take place at pH greater than 8.0. Since thiocyanate does not bind to LPO above this pH, the binding of thiocyanate to LPO is considered to be prerequisite for the oxidation of thiocyanate. Maximum inhibition of oxygen uptake by Streptococcus cremoris 972 bacteria was observed when hydrogen peroxide and thiocyanate were present in equimolar amounts and the pH was below 6.0.     

Intramolecular electron transfer between tyrosyl radical and cysteine residue inhibits tyrosine nitration and induces thiyl radical formation in model peptides treated with myeloperoxidase, H2O2, and NO2-: EPR SPIN trapping studies

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.     

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization,characterization, mechanism of nitration,and biological implications

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.     

Production of nitric oxide by human salivary peroxidase and by bovine lactoperoxidase

Peroxidases catalyze the oxidation of nitrite to nitrate in the presence of hydrogen peroxide. Two pathways may occur: one entailing the intermediate formation of NO(2) and the other implying the generation of peroxynitrite. The products of nitrite (NO(2) (-) ) oxidation by salivary peroxidase (SPO) and commercial bovine lactoperoxidase (LPO) are studied by utilizing an electrochemical assay that allows the direct, continuous monitoring of NO and/or NO(2) and by HPLC to assess nitrates at the end of the reaction. Dialyzed saliva and LPO, in the presence of H(2) O(2) , convert nitrite into nitrate and form some NO, with a molar ratio of 10(3) . In our experimental conditions, no NO(2) was detectable among the products of nitrite oxidation. SCN(-) inhibits NO formation and so does I(-) , although at higher concentrations. No effects are observed with Cl(-) or Br(-) . We conclude that SPO and LPO transform NO(2) (-) into nitrate-forming small amounts of NO in the presence of H(2) O(2) as an intermediate or a by-product, synthesized through the peroxynitrite pathway.     

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ～7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.     

A spectroscopic and voltammetric study of the pH-dependent Cu(II) coordination to the peptide GGGTH: relevance to the fifth Cu(II) site in the prion protein

The GGGTH sequence has been proposed to be the minimal sequence involved in the binding of a fifth Cu(II) ion in addition to the octarepeat region of the prion protein (PrP) which binds four Cu(II) ions. Coordination of Cu(II) by the N- and C-protected Ac-GGGTH-NH(2) pentapeptide (P(5)) was investigated by using potentiometric titration, electrospray ionization mass spectrometry, UV-vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry experiments. Four different Cu(II) complexes were identified and characterized as a function of pH. The Cu(II) binding mode switches from NO(3) to N(4) for pH values ranging from 6.0 to 10.0. Quasi-reversible reduction of the [Cu(II)(P(5))H(-2)] complex formed at pH 6.7 occurs at E (1/2)=0.04 V versus Ag/AgCl, whereas reversible oxidation of the [Cu(II)(P(5))H(-3)](-) complex formed at pH 10.0 occurs at E (1/2)=0.66 V versus Ag/AgCl. Comparison of our EPR data with those of the rSHaPrP(90-231) (Burns et al. in Biochemistry 42:6794-6803, 2003) strongly suggests an N(3)O binding mode at physiological pH for the fifth Cu(II) site in the protein.     

Genome-guided exploration of metabolic features of Streptomyces peucetius ATCC 27952: past,current, and prospect

Applied Microbiology and Biotechnology - Streptomyces peucetius ATCC 27952 produces two major anthracyclines, doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for...     

Biomimetic oxidation of ibuprofen with hydrogen peroxide catalysed by horseradish peroxidase (HRP) and 5,10,15,20-tetrakis-(2',6'-dichloro-3'-sulphonatophenyl)porphyrinatoiro n(III) and manganese(III) hydrates in AOT reverse micelles

The oxidation of ibuprofen with H2O2 catalysed by Horseradish peroxidase (HRP), Cl8TPPS4Fe(III)(OH2)2 and Cl8TPPS4Mn(III)(OH2)2 in AOT reverse micelles gives 2-(4'-isobutyl-phenyl)ethanol (5) and p-isobutyl acetophenone (6) in moderate yields. The reaction of ibuprofen (2) with H2O2 catalysed by HRP form carbon radicals by the oxidative decarboxylation, which on reaction with molecular oxygen to form hydroperoxy intermediate, responsible for the formation of the products 5 and 6. The yields of different oxidation products depend on the pH, the water to surfactant ratio (Wo), concentration of Cl8TPPS4Fe(III)(OH2)2 and Cl8TPPS4Mn(III)(OH2)2 and amount of molecular oxygen present in AOT reverse micelles. The formation of 2-(4'-isobutyl phenyl)ethanol (5) may be explained by the hydrogen abstraction from ibuprofen by high valent oxo-manganese(IV) radical cation, followed by decarboxylation and subsequent recombination of either free hydroxy radical or hydroxy iron(III)/manganese(III) porphyrins. The over-oxidation of 5 with high valent oxo-manganese, Mn(IV)radical cation intermediate form 6 in AOT reverse micelles by abstraction and recombination mechanism.     

Myeloperoxidase potentiates nitric oxide-mediated nitrosation

Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 mum) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 mum H(2)O(2)/min, and 0.024 mum NO/min. Autoxidation was only detected at >/=1.2 mum NO/min. MPO potentiation was not affected by a 40-fold excess flux of H(2)O(2) over NO or less than a 2.4-fold excess flux of NO over H(2)O(2). Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO(+). MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO(2)(.) or an NO(2)(.)-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO(2)(-) at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO(2)(-). Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN(3). This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration.     

The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen species: A self-fulfilling prophesy?

The oxidation of 2',7'-dichlorofluorescin (DCFH) and its diacetate form (DCFHDA) by the HRP/peroxynitrite system was investigated. Both DCFH and DCFHDA were oxidized to fluorescent products. A major anomaly, however, was the observation that fluorescence continued to build up long after peroxynitrite total decomposition and the initial HRP compound I reduction, suggesting the production of oxidants by the system. Indeed, preformed HRP compound I was instantly reduced by DCFH and DCFHDA to compound II with the obligate formation of DCF(-) semiquinone and DCFHDA-derived radicals. Catalase strongly inhibited fluorescence and EPR signals, suggesting the intermediate formation of H2O2. Taken together the data indicate that peroxynitrite rapidly oxidizes HRP to HRP compound I, which is reduced by DCFH and its diacetate form with the concomitant formation of DCF(-) semiquinone and DCFHDA-derived radicals. These are oxidized by O2, producing O2(-) (as demonstrated by EPR and oxygen consumption experiments), which dismutates to produce H2O2, which serves to fuel further DCFH/DCFHDA oxidation via HRP catalysis. Also DCFHDA was shown to be considerably more resistant to oxidation than its hydrolyzed product DCFH, presumably because of the absence of the easily oxidizable phenol moieties. DCFHDA/DCFH have been used to study free radical production in a variety of systems. Our findings demonstrate that this assay is subject to a serious artifact in that it produces what it is purported to measure; therefore, its use in biological systems should be approached with caution.     

Formation of nitrogen oxides and citrulline upon oxidation of N omega-hydroxy-L-arginine by hemeproteins.

HRP catalyzes the oxidation of N omega-hydroxy-L-arginine (NOHA) by H2O2 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases.     

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.     

Syntheses,characterization and X-ray structures of the fac-[RuCl3(NO)(dppe)] and the trans-[RuCl(NO)(dppe)2]2+ species

Trans-[RuCl(NO)(dppe)2]2+ species were prepared. The complexes have been characterized by microanalysis, IR and 31P[1H] NMR spectroscopy and cyclic voltammetry. The trans-[RuCl(NO)(dppe)2](ClO4)2 complex shows a reversible one-electron-reduction process at E(1/2) = 0.200 V and another one-electron-reduction irreversible process at -0.620 V, both centered at the NO+ group. The dissociation of the NO group from the trans-[RuCl(NO)(dppe)2]2+ after two one-electron reductions results in the formation of the trans- and cis-[RuCl2(dppe)2] isomers. The product of an electrolyzed solution of the same complex at -0.300 V shows an EPR signal consistent with the presence of the [RuCl(NO(0))(dppe)2]+ complex. Crystal data for trans-[RuCl(NO)(dppe)2]2+*[RuCl4(NO)(H2O)]*1/2[RuCl6]4-*2[H2O] (I) and trans-[RuCl(NO)(dppe)(2)]2+*2[RuCl4(NO)(CH3O)]-*3[CH3OH] (II) are as follow: (I) Space group P-1, a=10.4040(3) A, b=12.3470(4) A, c=23.5620(8) A, alpha=95.885(2) degrees, beta=99.608(2) degrees, gamma=104.378(2) degrees, R=0.0521; (II) space group P-1, a=10.9769(2) A, b=13.2753(3) A, c=24.0287(4) A, alpha=99.743(1) degrees, beta=95.847(1) degrees, gamma=97.549(1) degrees; R=0.0496. The fac-[RuCl3(NO)(dppe)] (III) complex has been also prepared; its crystal data are: space group P2(1)/n (No. 14), a=11.841(2) A, b=13.775(2) A, c=16.295(4) A, beta=92.81(2) degrees; R1=0.0395.     

EDTA inhibits peroxidase-catalyzed iodide oxidation through interaction at the iodide binding site

EDTA inhibits the formation of I3- from iodide catalysed by various pure peroxidases. The inhibition is concentration-dependent and chloroperoxidase (CPO) is more sensitive than horseradish peroxidase (HRP) and lactoperoxidase (LPO). EDTA is more active than EGTA or other biological chelators tested. Zn2+, Mn2+ and Co2+ are equally active in reversing the effect of EDTA on both CPO and HRP almost completely, but ineffective in the case of LPO. The effect of EDTA on HRP can be reversed by a higher concentration of iodide but not by H2O2. EDTA causes a hypsochromic change in the absorption of the Soret band of HRP at 402 nm, and iodide can reverse this effect. EDTA can effectively displace radioiodide specifically bound to HRP. It is suggested that EDTA inhibits iodide oxidation by interacting at the iodide binding site of the HRP.