Initial Oxidation Rate Research Articles

Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3

We recently reported conversion of cytochrome P450 BM-3, a medium-chain (C 12–C 18) fatty acid monooxygenase, into a highly efficient alkane hydroxylase by directed evolution [ Nat. Biotechnol. 2002, 20, 1135]. P450 BM-3 mutant 139-3 exhibited high activity towards a variety of fatty acid and alkane substrates, including C 3–C 8 alkanes. We report here that mutant 139-3 is also active on benzene, styrene, cyclohexene, 1-hexene, and propylene. Benzene is converted to phenol, while styrene is converted to styrene oxide. Propylene oxidation generates only propylene oxide, but cyclohexene oxidation produces a mixture of cyclohexene oxide (85%) and 2-cyclohexene-1-ol (15%), and 1-hexene is converted to the allylic hydroxylation product, 1-hexene-3-ol. Initial rates of NADPH oxidation for 139-3 in the presence of the substrates greatly (17- to >100-fold) surpass the wild-type in all cases. However, NADPH consumption is only partially coupled to product formation (14–79%). This cytochrome P450 epoxidation catalyst is a suitable starting point for further evolution to improve coupling and activity.

A Study on the Process of Lipase-catalyzed Synthesis of α-pinene Oxide in Organic Solvents

The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L−1 h−1 when the amount of H2O2 increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H2O2 for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.

Mono and oligonuclear vanadium complexes as catalysts for alkane oxidation: synthesis, molecular structure, and catalytic potential

A series of mono- and oligonuclear vanadium(V) and vanadium(IV) complexes containing various chelating N, O-, N 3-, and O 2-ligands have been prepared. The biphasic reaction of an aqueous solution of ammonium vanadate and a dichloromethane solution of hexamethylphosphoramide (hmpa) and pyrazine-2-carboxylic acid (pcaH) or pyrazine-2,5-dicarboxylic acid (pdcaH 2) or pyridine-2,5-dicarboxylic acid (pycaH 2) yields yellow crystals of [VO 2(pca)(hmpa)] ( 1), [(VO 2) 2(pdca)(hmpa) 2] ( 2), and [VO 2(pycaH)(hmpa)] ( 3), respectively. The single-crystal X-ray structure analyses reveal 1 and 3 to be mononuclear vanadium(V) complexes, in which a VO 2 unit coordinates to one nitrogen and one oxygen atom of a pca or pycaH chelating ligand, and 2 to be a dinuclear vanadium(V) complex, in which two VO 2 units are coordinated through one nitrogen and one oxygen atom of a pdca bridging ligand; in the three complexes the vanadium atoms also coordinate to the oxygen atom of a hmpa ligand. The reaction of N, N, N ′, N ′-tetrakis(2-benzimidazolylmethyl)-2-hydroxo-1,3-diaminopropane (hptbH) and VOSO 4 in methanol gives the cationic complex [(VO) 4(hptb) 2(μ-O)] 4+ ( 4), which can be crystallized as the perchlorate salt. In this tetranuclear complex, two dinuclear vanadium(IV) units are held together by a μ-oxo bridge. The known complex [VOCl 2(tmtacn)] ( 5) was synthesized from the reaction of 1,4,7-trimethyl-1,4,7-triazacyclononane (tmtacn) and VCl 3 in acetonitrile; the reaction of tetrabutylammonium vanadate with pyro-cathecol (catH 2) in acetonitrile gives the known anionic complex [V(cat) 3] − ( 6), in which the vanadium(V) center is bonded to three cat chelating ligands through the oxygen atoms, obtained as the tetrabutylammonium salt. All compounds synthesized are highly efficient oxidation catalysts for the reaction of cyclohexane with air and hydrogen peroxide in the presence of four equivalents of pcaH per vanadium, although the catalytic activity of the complexes containing bulky chelating ligands 4 and 5 is somewhat lower in the initial period of the reaction. During this period the active species are formed from the complexes and final turnover numbers are high. The catecholate ligands of complex 6 may reduce from V(V) to V(IV) in the beginning of the process, thus providing very high initial oxidation rates.

Synthesis, crystal structure and spectral studies of Cu(II)/Cu(I) complexes derived from diamide bisbenzimidazole ligand

A new benzimidazole based diamide ligand— N, N′-bis(2-methylbenzimidazolyl)-2,2′-oxydiethanamide (GBOA)—has been synthesized and utilized to prepare [Cu(GBOA)Cl 2] and [Cu I(NO 3)(GBOA)]. The complex [Cu(GBOA)Cl 2] has been characterized structurally. It crystallizes in monoclinic space group P2 1/ n. The Cu(II) ion is in a slightly distorted square pyramidal coordination environment ( τ=0.167). Two trans bonded chlorine atoms, and two nitrogen atoms from two different molecules of benzimidazole ligand form the basal plane while the amide oxygen occupies the axial position of the square based pyramid. The structure of the complex is a one-dimensional, ligand-bridged polymer. Solid state X-band EPR spectrum of the complex shows a slightly broadened isotropic signal at g=2.0. The complexes display a quasi-reversible redox wave due to the Cu(II)/Cu(I) redox couple with E 1/2 values varying as NO 3 − <Cl −. With respect to NHE, the E 1 2 values are quite positive when compared to other benzimidazole based complexes (Inorg. Chim. Acta 151 (1988) 55). The catechol oxidase activity of [CuCl 2(GBOA)] has been examined by studying the oxidation of various catechols in oxygen saturated methanol. Kinetic studies with [CuCl 2(GBOA)] and DTBC (3,5-di- tert-butylcatechol) as substrate shows the initial rate of oxidation to be dependent on first power of catalyst concentration and square-root of substrate concentration.

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic α,β-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST–4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 μM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets.

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.

Effects of N‐fertilisation on CH4 oxidation and production, and consequences for CH4 emissions from microcosms and rice fields

AbstractThe world's growing human population causes an increasing demand for food, of which rice is one of the most important sources. In rice production nitrogen is often a limiting factor. As a consequence increasing amounts of fertiliser will have to be applied to maximise yields. There is an ongoing discussion on the possible effects of fertilisation on CH4 emissions. We therefore investigated the effects of N‐fertiliser (urea) on CH4 emission, production and oxidation in rice microcosms and field experiments. In the microcosms, a substantial but short‐lived reduction of CH4 emission was observed after N‐addition to 43‐d‐old rice plants. Methane oxidation increased by 45%, demonstrated with inhibitor measurements and model calculations based on stable carbon isotope data (δ13CH4). A second fertilisation applied to 92‐d‐old plants had no effect on CH4 emission rates.The positive effect of additional N on methanotrophic bacteria was also found in vitro for potential CH4 oxidation rates in soil and root samples from the microcosm and field experiments, indicated by elevated initial oxidation rates and reduced lag‐phases. Fertilisation did not affect methane production in the microcosms. In the field, the effects were diverse: methane production was inhibited in the topsoil, but stimulated instead in the bulk soil. Stimulation occurred probably in the anaerobic food chain at the level of hydrolytic or fermenting bacteria, because acetate, a methanogenic precursor, increased simultaneously.Combining field, microcosm and laboratory experiments we conclude that any agricultural treatment improving the N‐supply to the rice plants will also be favourable for the CH4 oxidising bacteria. However, N‐fertilisation had only a transient influence and was counter‐balanced in the field by an elevated CH4 production. A negative effect of the fertilisation was a transient increase of N2O emissions from the microcosms. However, integrating over the season the global warming potential (GWP) of N2O emitted after fertilisation was still negligible compared to the GWP of emitted CH4.

Laccase-catalyzed oxidation of naphthol in the presence of soluble polymers

The oxidation of 1-naphthol and 2-naphthol was studied in presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL). During 1-naphthol oxidation a violet colored insoluble product was formed. The product of 2-naphthol oxidation was a white insoluble precipitate. Almost two moles of 1-naphthol was oxidized by one mole of oxygen. Kinetic measurements of oxygen consumption showed that laccases have been inactivated during naphthols oxidation. Water soluble polymers and albumins changed oxidation profile. The polymers investigated were divided into three groups. The first group includes polymers which increase total oxygen consumption without changing an initial rate: polyvinyl alcohol, ficoll, a copolymer of acrylamide, N-vinylpyrrolidone and ethyl acrylate, hydroxyethyl cellulose, dextrans 500, 110 and 20, polyvinylpyrrolidone, polyethylene glycol and albumins. These polymers intermediately bound hydrophobic naphtholic radicals preventing inactivation of laccases. Cationic polymers such as diethylaminoethyl-dextran, poly- l-lysine and protasan that inhibited the initial oxidation rate, but did not change total oxygen consumption, were attributed to the second group. The inhibition was associated with complexation of positively charged polymers with negatively charged laccases since isoelectric points of rPpL and of rMtL were 3.5 and 4.2, respectively. The polymers such as alginic acid, polyacrylic acid and heparin were attributed to the third group. These polymers carried negative charges and formed random coil in solution due to electrostatic repulsion. They did not interact with laccases and did not combine intermediates. Therefore, they practically did not change total oxygen consumption and initial rate of naphthol oxidation.

Antioxidant and Prooxidant Effects of Tocopherol

A computer simulation of the kinetics of free-radical oxidation of lipids in the presence of tocopherol was performed. The induction periods and initial rates of oxidation as functions of initial tocopherol concentrations were found to exhibit extremums. The extended kinetic scheme used in this study describes all types of observed relationships and eliminates illusory contradictions between two groups of experiments in which the antioxidant and prooxidant effects of tocopherol were found. The model can form a basis for the prediction of the effects of tocopherol and its analogs under real conditions.

In situ surface oxidation of Ti-44Al-11Nb alloy at room temperature

The in situ surface oxidation of polycrystalline Ti-44Al-11Nb (compositions are in atomic %) alloy was studied at room temperature in an ultrahigh vacuum (66.6 to 80.0 nPa). The native oxide of Ti-44Al-11Nb was removed by sputtering the surface using 2.8 kV argon ions and then high-purity oxygen was carefully dosed onto the aluminide surface. After each oxygen dosing the surface species, metallic states and oxidized states, were detected by X-ray photoelectron spectroscopy (XPS). After analyzing the binding energy shifts and the concentration of oxidized species, aluminum oxidized first, then titanium followed by niobium. A reaction sequence model, incorporating the rate-determining step of a dissociative adsorption of oxygen, shows to be consistent with the initial oxidation rate of the Ti-44Al-11Nb intermetallic.

Oxidation activity and stability of homogeneous cobalt-sulphosalen catalyst: Studies with a phenolic and a non-phenolic lignin model compound in aqueous alkaline medium

Oxidation activity and stability of cobalt-Schiff base complex catalyst, Co-sulphosalen, were studied in aqueous alkaline medium. The 4-coordinated Co-sulphosalen was shown to be an active catalyst, with molecular oxygen increasing the initial oxidation rates of both a phenolic (guaiacol) and a non-phenolic (veratryl alcohol) lignin model compounds. Studies with a visible absorption spectrometer showed that 4-coordinated Co-sulphosalen forms a new complex with pyridine, which attaches as axial ligand. The activity of Co-sulphosalen complex was not increased, however, but instead was slightly decreased by the addition of pyridine. With a newly developed HPLC method, it was shown that Co-sulphosalen is not stable but decomposes as a function of time. The decomposition of the catalyst through hydrolysis of its imine structures was independent of the oxidation of the model compound. The decomposition rate of Co-sulphosalen increased with increasing pH and was higher in the absence of oxygen. The presence of pyridine had virtually no effect on the stability of the catalyst.

Preparation of Nanosize Anatase and Rutile TiO2 by Hydrothermal Treatment of Microemulsions and Their Activity for Photocatalytic Wet Oxidation of Phenol

Titanium dioxide (TiO2) nanoparticles of both anatase and rutile phases were synthesized by hydrothermal treatment of microemulsions, and their photocatalytic activity for wet oxidation of phenol was studied. The only difference between the two syntheses used was that different acids were added to the microemulsions, making direct comparison of the catalytic activity of the two polymorphs possible. If hydrochloric acid was used, the rutile structure formed, and if nitric acid was used, anatase formed. The phase stability of the microemulsion was studied and according to conductivity and turbidity measurements the idea of a direct template effect could be discarded during the hydrothermal treatment. However, an initial size-templating phenomenon is possible during the mixing step. The particles, which were in the size range of a few nanometers were characterized with N2-adsorption, XRD, SEM, and XPS. The activity of the two polymorphs for the photocatalytic oxidation of phenol in water was examined. It was shown that the rutile phase initially decomposed phenol much faster and follows a first-order process reasonably well (k = 4 × 10-5 s-1). The photodecomposition process using the anatase phase led, however, to a much more rapid overall degradation following an initial slower rate of phenol oxidation. The results indicate that the observed difference of the photodecomposition process for the two TiO2 phases is due to the formation of different intermediates.

Si-rich6H- and4H−SiC(0001)3×3 surface oxidation and initialSiO2/SiCinterface formation from 25 to 650 °C

We investigate the initial oxidation of the Si-rich $6H\ensuremath{-}\mathrm{SiC}(0001)$ 3\ifmmode\times\else\texttimes\fi{}3 and $4H\ensuremath{-}\mathrm{SiC}(0001)$ 3\ifmmode\times\else\texttimes\fi{}3 surfaces and the subsequent ${\mathrm{SiO}}_{2}/4H\ensuremath{-}\mathrm{SiC}$ and ${\mathrm{SiO}}_{2}/6H\ensuremath{-}\mathrm{SiC}$ initial interface formation by Si 2p, C 1s, and O 1s core-level photoemission spectroscopy using synchrotron radiation. The 3\ifmmode\times\else\texttimes\fi{}3 surface reconstruction is found to be highly reactive to oxygen with an initial oxidation rate of about three to four orders of magnitude larger than for silicon surfaces. Furthermore, for both polytypes, direct ${\mathrm{SiO}}_{2}/\mathrm{SiC}$ interface formation is achieved already at room temperature and extremely low oxygen exposures. However, the two polytypes have significantly different behaviors with larger amounts of oxide products having higher oxidation states for the $6H\ensuremath{-}\mathrm{SiC}(0001)$ 3\ifmmode\times\else\texttimes\fi{}3 surface, while mixed oxides including carbon species (Si-O-C) are the dominant oxide products for the 4H polytype surface. The oxidation rate is improved at increased surface temperatures. In all cases, the oxygen uptake remains significantly larger for the 6H polytype when compared to the 4H one. The very different behavior of the 6H and 4H polytypes seems to originate, at least in part, from the presence of two domains in the bulk for the 4H polytype (as evidenced by two bulk components in the Si 2p core-level spectrum) which limits the oxygen insertion into the $4H\ensuremath{-}\mathrm{SiC}$ lattice. Instead the 6H polytype has only one bulk domain with a single Si 2p bulk component. Abrupt ${\mathrm{SiO}}_{2}/6H\ensuremath{-}\mathrm{SiC}$ interfaces could be achieved by thermal oxidation of a predeposited Si overlayer onto the surface leading to have oxide thicknesses ranging from 10 \AA{} up to 80 \AA{} after postoxidation with a transition layer of less than 5 \AA{}. This study shows that, using a ``gentle'' initial oxidation approach at low temperatures and low oxygen exposures allow high-quality ${\mathrm{SiO}}_{2}/\mathrm{SiC}$ interfaces. It also brings interesting insights into the understanding of polytype crucial effect in SiC surface oxidation.

Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid: contribution to placental endothelial nitric oxide synthase activity.

The aim of this study was to characterize the mechanism of the chemical interaction between L-ascorbic acid (ASC) and tetrahydrobiopterin (BH(4)) in vitro and to examine its effect on the activity of endothelial nitric oxide synthase (eNOS) in first trimester human placentae. At room temperature, in Tris-HCl buffer (pH 7.4), both ASC and BH(4) were readily oxidized by dissolved O(2) or H(2)O(2). BH(4) was more sensitive to auto-oxidation, while ASC was more susceptible to oxidation by H(2)O(2). Addition of 36 micromol/l BH(4) to 143 micromol/l ASC increased the initial rate of ASC oxidation 3.2-fold in a catalase-sensitive manner, indicating that enhanced ASC oxidation is partly due to the formation of H(2)O(2). In the presence of catalase, BH(4) still stimulated 1.9-fold the initial rate of ASC oxidation, suggesting that another auto-oxidation product of BH(4), most probably quininoid-BH(2) (qBH(2)), could also stimulate ASC oxidation while itself being reduced back to BH(4). ASC prevented the auto-oxidation of BH(4) in a concentration-dependent fashion, with 3 mmol/l ASC providing an almost complete stabilization of 25 micromol/l BH(4). Importantly, basal eNOS activity in placental microsomes was stimulated 2.5-fold by 0.5 micromol/l BH(4), and 0.5 mmol/l ASC enhanced the BH(4)-stimulation 1.4-fold, with a smaller effect on basal eNOS activity. Taken together, the findings support the notion that the stabilizing action of ASC on BH(4) is related to the ASC-mediated reductive reversal of the auto-oxidation process of BH(4). Moreover, we demonstrated that concentrations of ASC present in the placenta as a common vitamin C supply are sufficient to protect cellular free BH(4) and may contribute to the stimulation of placental eNOS activity.

Properties of the GaAs oxide layer grown by the liquid-phase chemical-enhanced technique on the S-passivated GaAs surface

The properties of GaAs oxide layers prepared by liquid-phase chemical-enhanced oxidation of the (NH4)2Sx-passivated GaAs surface are investigated. The initial oxidation rate is suppressed and the refractive indices of oxide layers are lower after S passivation. In accordance with the depth profiles measured by secondary ion mass spectroscopy (SIMS), a model is proposed to illustrate the chemical composition of the oxide layer grown by liquid-phase chemical-enhanced oxidation after S passivation. Oxidation following different surface treatments was studied by analyzing their activation energies. In addition, we find that the leakage current and the breakdown electric field of oxide layers can be improved significantly with the passivation technique.

Influence of gas transport on the oxidation rate of aluminum arsenide

The effect of gas transport on the lateral oxidation kinetics of aluminum arsenide has been studied by limiting the gas transport to the gas–oxide interface using closely spaced mesas. At 440 °C the gas transport factor is found to be 8.5±1.3 times the reaction rate coefficient, resulting in a measurable decrease in oxidation rate for mesas closer than 200 nm. This confirms the usual assumption that the initial oxidation rate is limited by the reaction kinetics at the oxidizing interface and not the gas transport for normal oxidation conditions

Kinetic evidence for the identical nature of active centers for partial and deep oxidation of ethylene on silver

Kinetics of interaction of ethylene with pre-adsorbed oxygen on a silver film was studied at 473 K and different initial values of surface coverage with oxygen. Dependencies of the initial rates of partial and deep ethylene oxidation on the oxygen surface coverage manifest themselves as peaked curves with coinciding maxima. The results are considered as evidence that the processes of partial and deep ethylene oxidation on silver, when they occur at optimal conditions, proceed viaidentical active centers.

The oxidation of hydroquinone to p-benzoquinone catalysed by Cu(II) ions immobilized on acrylic resins with aminoguanidyl groups: Part 1

The immobilized macromolecular copper catalysts were prepared by modification of nitrile groups in acrylonitrile, vinyl acetate and divinylbenzene terpolymer using aminoguanidine carbonate and subsequent complexation with copper(II) nitrate. The activity of the catalysts was measured in the model reaction—oxidation of hydroquinone (H 2Q) to p-benzoquinone (Q) by hydrogen peroxide (H 2O 2). Under the optimal conditions only main product p-benzoquinone (yield 98%) was obtained after 80 min. The catalytic activity and selectivity in the Cu(II)–resin system increases compared to reaction without catalyst and with native Cu(II) ions. For the best catalyst, after 10 min, initial oxidation rate increased 57 times in comparison with the same reaction without catalyst as well as 16 times in comparison with native Cu(II) ions. The oxidation reaction of H 2Q showed a kinetic behaviour typical for a Michaelis–Menten mechanism. The optimal oxidation parameters in the reaction H 2Q→Q are (a) H 2Q:Cu(II) ratio—10:1, (b) H 2O 2 concentration—5×10 (mol/l) and (c) solution pH above 5.

Ceruloplasmin as low-density lipoprotein oxidase: activation by ascorbate and dehydroascorbate

The ability of ceruloplasmin (Cp) to oxidize low-density lipoproteins (LDL) in the presence of water-soluble antioxidants was investigated and a reaction mechanism proposed. Ascorbate strongly enhanced LDL oxidation, but only after its rapid consumption. Dehydroascorbate enhanced Cp-mediated LDL oxidation even more strongly. Lipid-soluble antioxidants and water-soluble peroxides did not show noticeable activation. However, loading of LDL with lipid hydroperoxides increased the initial oxidation rate. We conclude that Cp mediates a localized redox cycle, where reduction of Cp-Cu 2+ is effected by water-soluble reductants and reoxidation by liposoluble hydroperoxides.

Chimeras of Nitric-oxide Synthase Types I and III Establish Fundamental Correlates between Heme Reduction, Heme-NO Complex Formation, and Catalytic Activity

Neuronal nitric-oxide synthase (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) differ widely in their reductase and nitric oxide (NO) synthesis activities, electron transfer rates, and propensities to form a heme-NO complex during catalysis. We generated chimeras by swapping eNOS and nNOS oxygenase domains to understand the basis for these differences and to identify structural elements that determine their catalytic behaviors. Swapping oxygenase domains did not alter domain-specific catalytic functions (cytochrome c reduction or H(2)O(2)-supported N(omega)-hydroxy-l-arginine oxidation) but markedly affected steady-state NO synthesis and NADPH oxidation compared with native eNOS and nNOS. Stopped-flow analysis showed that reductase domains either maintained (nNOS) or slightly exceeded (eNOS) their native rates of heme reduction in each chimera. Heme reduction rates were found to correlate with the initial rates of NADPH oxidation and heme-NO complex formation, with the percentage of heme-NO complex attained during the steady state, and with NO synthesis activity. Oxygenase domain identity influenced these parameters to a lesser degree. We conclude: 1) Heme reduction rates in nNOS and eNOS are controlled primarily by their reductase domains and are almost independent of oxygenase domain identity. 2) Heme reduction rate is the dominant parameter controlling the kinetics and extent of heme-NO complex formation in both eNOS and nNOS, and thus it determines to what degree heme-NO complex formation influences their steady-state NO synthesis, whereas oxygenase domains provide minor but important influences. 3) General principles that relate heme reduction rate, heme-NO complex formation, and NO synthesis are not specific for nNOS but apply to eNOS as well.