Fast Reoxidation Research Articles

Conductive supports for combined AFM–SECM on biological membranes

Four different conductive supports are analysed regarding their suitability forcombined atomic force and scanning electrochemical microscopy (AFM–SECM)on biological membranes. Highly oriented pyrolytic graphite (HOPG),MoS2, template stripped gold, and template stripped platinum are compared as supports forhigh resolution imaging of reconstituted membrane proteins or native membranes, and aselectrodes for transferring electrons from or to a redox molecule. We demonstrate that highresolution topographs of the bacterial outer membrane protein F can be recorded by contactmode AFM on all four supports. Electrochemical feedback experiments with conductivecantilevers that feature nanometre-scale electrodes showed fast re-oxidation of the redox coupleRu(NH3)63+/2+ with the two metal supports after prolonged immersion in electrolyte. In contrast,the re-oxidation rates decayed quickly to unpractical levels with HOPG orMoS2 under physiological conditions. On HOPG we observed heterogeneity in the re-oxidationrate of the redox molecules with higher feedback currents at step edges. Thelatter results demonstrate the capability of conductive cantilevers with smallelectrodes to measure minor variations in an SECM signal and to relate themto nanometre-scale features in a simultaneously recorded AFM topography.Rapid decay of re-oxidation rate and surface heterogeneity make HOPG orMoS2 less attractive for combined AFM–SECM experiments on biological membranes thantemplate stripped gold or platinum supports.

Ferrocyanide − a novel catalyst for oxymyoglobin oxidation by molecular oxygen

A comparative study of the rates of ferrocyanide-catalyzed oxidation of several oxymyoglobins by molecular oxygen is reported. Oxidation of the native oxymyoglobins from sperm whale, horse and pig, as well as the chemically modified (MbO(2)) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM-MbO(2)), and the mutant sperm whale oxymyoglobin [MbO(2)(His119-->Asp)], was studied. The effect of pH, ionic strength and the concentration of anionic catalyst ferrocyanide, [Fe(CN)(6)](4-), on the oxidation rate is investigated, as well as the effect of MbO(2) complexing with redox-inactive Zn(2+), which forms the stable chelate complex with functional groups of His119, Lys16 and Asp122, all located nearby. The catalytic mechanism was demonstrated to involve specific [Fe(CN)(6)](4-) binding to the protein in the His119 region, which agrees with a high local positive electrostatic potential and the presence of a cavity large enough to accommodate [Fe(CN)(6)](4-) in that region. The protonation of the nearby His113 and especially His116 plays a very important role in the catalysis, accelerating the oxidation rate of bound [Fe(CN)(6)](4-) by dissolved oxygen. The simultaneous occurrence of both these factors (i.e. specific binding of [Fe(CN)(6)](4-) to the protein and its fast reoxidation by oxygen) is necessary for the efficient ferrocyanide-catalyzed oxidation of oxymyoglobin.

High catalytic activity and stability of Pd doped hexaaluminate catalysts for the CH4 catalytic combustion

In this work, different procedures, namely carbonate coprecipitation and modified solid–solid diffusion, were used to prepare hexaaluminate samples, unsupported or supported onto θ-Al2O3. These samples were used as catalyst for the methane total oxidation as synthesized or after impregnation of 1wt% Pd. It was observed that the modified solid–solid diffusion procedure is an efficient method to obtain the hexaaluminate structure. At a theoretical ratio x of hexaaluminate onto Al2O3 less than 0.6 (xLa0.2Sr0.3Ba0.5MnAl11O19+(1−x)·Al2O3, with x=0.25, 0.60), samples with high specific surface area and θ-Al2O3 structure are then obtained. Large differences in catalytic activity can be observed among the series of sample synthesized. All the pure oxide samples (i.e. without palladium) present low catalytic activity for methane total oxidation compared to a reference Pd/Al2O3 catalyst. The highest activity was obtained for the samples presenting a θ-Al2O3 structure (with x=0.60) and a high surface area. Impregnation of 1wt% palladium resulted in an increase in catalytic activity, for all the solids synthesized in this work. Even if the lowest light-off temperature was obtained on the reference sample, similar methane conversions at high temperature (700°C) were obtained on the stabilized θ-Al2O3 solids (x=0.25, 0.60). Moreover, the reference sample is found to strongly deactivate with reaction time at the temperature of test (700°C), due to a progressive reduction of the PdOx active phase into the less active Pd° phase, whereas excellent stabilities in reaction were obtained on the pure and palladium-doped hexaaluminate and supported θ-Al2O3 samples. This clearly showed the beneficial effect of the support for the stabilization of the PdOx active phase at high reaction temperature. These properties are discussed in term of oxygen transfer from the support to the palladium particle. Oxygen transfer is directly related to the Mn3+/Mn2+ redox properties (in the case of the hexaaluminate and stabilized θ-Al2O3 samples), that allows a fast reoxidation of the metal palladium sites since palladium sites reoxidation cannot occur directly by gaseous dioxygen adsorption and dissociation on the surface.

Oxidative Dehydrogenation of Propane by Monomeric Vanadium Oxide Sites on Silica Support

We investigate possible mechanisms of oxidative dehydrogenation of propane using density functional theory. Monomeric vanadium oxide species supported on silica are modeled by vanadyl-substituted silsesquioxane. Similarly to other catalysts with transition metal oxo bonds, the initial C−H bond activation step is hydrogen abstraction by the vanadyl (OVV) group yielding a diradical intermediate in which a propyl radical is bound to a HO−VIV site. This is followed by a propyl rebound mechanism yielding alkoxide or alcohol attached to a VIII(OSi)3 surface site from which propene can be formed. Propene is also directly obtained by a second hydrogen abstraction from the diradical intermediate. Desorption of propyl radicals leads to a stationary concentration of propyl in the gas phase and leaves reduced HO−VIV sites on the surface. Due to fast reoxidation their concentration is much smaller than the concentration of OVV sites. Therefore the rate of propene formation after readsorption on OVV sites is much large...

A dip in the chlorophyll fluorescence induction at 0.2–2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2–2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100–1800 μmol photons m −2 s −1). Simultaneous measurements of FI and 820-nm transmission kinetics (I 820) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2–0.4 s) is accompanied by a decrease of I 820, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4–2 s) of the dip is not paralleled by the change in I 820. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I–P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I 820 kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin–NADP +–oxidoreductase or Mehler–peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.

Light-induced proton slip and proton leak at the thylakoid membrane

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H+/e(-) -ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF1-gamma of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF1-gamma was only 36%, which indicates that in vivo the reduction of CF1-gamma could be limited by fast reoxidation and/or restricted accessibility of CF1-gamma to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.

Formation of Cu(I) in estuarine and marine waters: application of a new solid-phase extraction method to measure Cu(I).

Cu(II) is a key species with respect to the bioavailability and hence toxicity of copper. Therefore, it is important to elucidate the factors that control Cu(I) steady-state concentrations in natural waters. In this study, a solid-phase-extraction-based method was developed that allows Cu(I) measurements at ambient concentrations. Cu(I) is selectively enriched as a bathocuproine complex on a hydrophobic polymer column, whereas Cu(II), bound to ethylenediamine, is not retained on the column. After elution with acidic methanol, Cu is analyzed with graphite-furnace atomic absorption spectroscopy. The detection limit of the whole analytical procedure is below 1 x 10(-9) M, and the mean recovery of Cu(I) is approximately 70%. We then applied this method to determine Cu(I) in water samples collected from the River Scheldt estuary and the North Sea. Upon irradiation of these filtered water samples in the laboratory (with approximately 5 kW m(-2)), Cu(I) steady-state concentrations ([Cu(I)]ss) were established within a few minutes, and [Cu(I)]ss ranged from 5% to 80% of total dissolved copper, depending on the origin of the water samples. Measured [Cu(I)]ss can be interpreted by considering light-induced reduction of Cu(II) and stabilization of Cu(I) by chloride at high salinity, thermal reduction of Cu(II) by sulfide-containing compounds at low salinity, and fast reoxidation of Cu(I) due to stabilization of Cu(II) by strong organic ligands present at intermediate salinity.

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn 2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation

The effect of Zn 2+ on the rates of electron transfer and of voltage generation in the cytochrome bc 1 complex ( bc 1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn 2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561–570 nm) the reduction of its high potential heme b h (in the absence of Zn 2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c 1 reduction (as monitored at 552–570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn 2+ <100 μM the reduction of heme b h remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc 1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b h is the main electrogenic reaction in the cytochrome bc 1 complex, (2) imply an allosteric link between the site of heme b h oxidation and the site of cytochrome c 1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc 1 complex.

Degradation of Sucrose and Nitrate Over Titania Coated Nano-hematite Photocatalysts

Photodegradation of sucrose and/or nitrate in aqueous solutions was studied over the titania coated nano-hematite photocatalysts under near-UV irradiation. The efficiency of the photocatalytic oxidation and reduction of these coated particles was compared to the prepared single phase TiO2. It was found that in different environments (O2 or N2) the particles showed differences in the photocatalytic efficiency due to the different reaction mechanisms. Effect of nitrate on sucrose degradation was investigated. In aerobic conditions, sucrose was degraded effectively while nitrate reduction was insignificant due to the fast reoxidation of nitrite by O2 in the dark and by OH. in homogeneous reaction. Since nitrite competes with sucrose for hydroxyl radicals, it has an adverse effect when the aerobic system contains both substrates. Since reoxidation was suppressed in N2 conditions, greater reduction of nitrate was observed. The result showed clearly that in the absence of O2, CO2 production from sucrose mineralisation was limited by the amount of oxygen produced from the nitrate decomposition. Partial photodissolution of Fe ions for the coated samples was also observed in both environments.

Onset of resistive internal interfaces in SrTi0.95Nb0.05O3+δ materials on changing from reducing to oxidising conditions and on cooling

Impedance spectroscopy has been used to investigate the long-term drift of the electrical behaviour of SrTi0.95Nb0.05O3+δ materials. The behavior of dense samples revealed the onset of resistive internal interfaces on cooling below about 950 °C in oxidizing conditions (typically air or N2). Dense materials repond very slowly to step changes from reducing conditions to air; this was monitored by impedance spectroscopy to separate the contributions of the bulk and of internal interfaces. After an initial stage the resistance of internal interfaces increases slowly and almost linearly with time for up to three weeks. The changes in bulk resistance are smaller, and tend to level out after a smaller time interval, possibly because the reoxidation of the sample is very sluggish. Porous samples show relatively fast reoxidation, and thus a fast increase in bulk resistance.

Tyrosinase Models. Synthesis, Structure, Catechol Oxidase Activity, and Phenol Monooxygenase Activity of a Dinuclear Copper Complex Derived from a Triamino Pentabenzimidazole Ligand.

The dicopper(II) complex with the ligand N,N,N',N',N"-pentakis[(1-methyl-2-benzimidazolyl)methyl]dipropylenetriamine (LB5) has been synthesized and structurally characterized. The small size and the quality of the single crystal required that data be collected using synchrotron radiation at 276 K. [Cu(2)(LB5)(H(2)O)(2)][ClO(4)](4): platelet shaped, P&onemacr;, a = 11.028 Å, b = 17.915 Å, c = 20.745 Å, alpha = 107.44 degrees, beta = 101.56 degrees, gamma = 104.89 degrees, V = 3603.7 Å(3), Z = 2; number of unique data, I >/= 2sigma(I) = 3447; number of refined parameters = 428; R = 0.12. The ligand binds the two coppers nonsymmetrically; Cu1 is coordinated through five N donors and Cu2 through the remaining three N donors, while two water molecules complete the coordination sphere. Cu1 has distorted TBP geometry, while Cu2 has distorted SP geometry. Voltammetric experiments show quasireversible reductions at the two copper centers, with redox potential higher for the CuN(3) center (0.40 V) and lower for the CuN(5) center (0.17 V). The complex binds azide in the terminal mode at the CuN(3) center with affinity lower than that exhibited by related dinuclear polyaminobenzimidazole complexes where this ligand is bound in the bridging mode. The catechol oxidase activity of [Cu(2)(LB5)](4+) has been examined in comparison with that exhibited by [Cu(2)(L-55)](4+) (L-55 = alpha,alpha'-bis{bis[(1-methyl-2-benzimidazolyl)methyl]amino}-m-xylene) and [Cu(2)(L-66)](4+) (L-66 = alpha,alpha'-bis{bis[2-(1-methyl-2-benzimidazolyl)ethyl]amino}-m-xylene) by studying the catalytic oxidation of 3,5-di-tert-butylcatechol in methanol/aqueous buffer pH 5.1. Kinetic experiments show that [Cu(2)(L-55)](4+) is the most efficient catalyst (rate constant 140 M(-1) s(-1)), followed by [Cu(2)(LB5)](4+) (60 M(-1) s(-1)), in this oxidation, while [Cu(2)(L-66)](4+) undergoes an extremely fast stoichiometric phase followed by a slow and substrate-concentration-independent catalytic phase. The catalytic activity of [Cu(2)(L-66)](4+), however, is strongly promoted by hydrogen peroxide, because this oxidant allows a fast reoxidation of the dicopper(I) complex during turnover. The activity of [Cu(2)(LB5)](4+) is also promoted by hydrogen peroxide, while that of [Cu(2)(L-55)](4+) is little affected. The phenol monooxygenase activity of [Cu(2)(LB5)](2+) has been compared with that of [Cu(2)(L-55)](2+) and [Cu(2)(L-66)](2+) by studying the ortho hydroxylation of methyl 4-hydroxybenzoate to give methyl 3,4-dihydroxybenzoate. The LB5 complex is much more selective than the other complexes since its reaction produces only catechol, while the main product obtained with the other complexes is an addition product containing a phenol residue condensed at ring position 2 of the catechol.

Inhibition of CO2-fixation and its effect on the activity of Photosystem II, on D1-protein synthesis and phosphorylation

To study the effects of limitations in the Calvin-cycle on Photosystem (PS) II function and on its repair by D1-protein turnover, glycerinaldehyde (DLGA) was applied to 1 h dark-adapted pea leaves via the petiole. The application resulted in a 90% inhibition of photosynthetic oxygen evolution after 90 min illumination at either 120 or 500 µmol m−2 s−1. In the control leaves an increase of light-dependent oxygen production to 147 and 171% was observed after 90 min illumination. According to chlorophyll fluorescence quenching analysis the inhibition of photosynthetic electron transport by DLGA led to a substantial increase in the reduction state of the primary quinone acceptor of PS II, QA, and to a rise in membrane energetisation. However, PS II functionality was hardly affected by DLGA at the low light intensity as indicated by the constant high yield of variable fluorescence, Fv/Fm. Only at 500 µmol m−2 s−1 a 15% loss of Fv/Fm was observed in the presence of DLGA indicating that inactivated PS II centres had accumulated. The control leaves also showed a slight loss of Fv/Fm which did not affect photosynthetic electron transport due to a faster reoxidation of QA. The relative stability of PS II function in the presence of DLGA could not be ascribed to an increased repair by the rapid turnover of the D1-protein. Radioactive pulse-labelling studies with [14C] leucine in combination with immunological determination of the protein content revealed that both synthesis and degradation of the protein were inhibited in DLGA-treated leaves whereas in the control leaves a stimulation of D1-protein turnover was observed. The changes of D1-protein turnover could be explained by differences in the occupancy state of the QB-binding niche. A relation between the phosphorylation status of the PS II polypeptides and the turnover of the D1-protein could not be established. As shown by radioactive labelling with [32P]i, addition of DLGA led to an increase in the phosphorylation level of the PS II polypeptides D1 and D2 at the low light intensity when compared to the non-treated control. At the higher light intensity the phosphorylation level of the PS II polypeptides in control and DLGA-treated leaves were identical in spite of the substantial differences in D1-protein turnover.

Improvement of High Temperature Water Rinsing and Drying for HF‐Last Wafer Cleaning

The reactions on HF cleaned (100)Si surfaces in high temperature water solutions are investigated by multiple internal reflection, Fourier transform infrared spectroscopy, atomic force microscopy, contact angle, and light scattering measurements. The hot water immersion causes a fast reoxidation, while a prolonged boiling water treatment results in an oxide‐free Si surface. It is found that the continuous gas bubbling can effectively drive the dissolved oxygen out of the boiling water. Comparing with the room temperature water rinsing, the boiling water treatment enhances the anisotropic etching on the HF cleaned bare silicon surface; and thus increases the microroughness on the Si(100) surface. The increased concentration in boiling water is found to be the main cause of the surface etching. So, the etching reaction can be effectively suppressed by adding HCl to control the concentration.

Photochemically induced redox reactions in seawater, I. Cations

Experiments have been conducted to determine whether the speciation of the manganese and iron redox couples can be influenced by light and organic matter in seawater. The results indicate that photochemically induced reduction of Mn(IV) to Mn(II) occurs through the mediation of humic acids. The rate of the reaction is dependent on many variables, some of which are identified here. Stirring plays an important role in the reductive dissolution process, the effect of which is dependent on the organic loading. It is suggested that this results from an adsorptive competition between organic matter and photoproduced reductants such as H 2 O 2 . Photochemically induced reduction reaction, at least in part, controls the speciation and thus biological availability of manganese in surface seawater. The results obtained from studies of the iron redox system show generally poor reproducibility and no conclusive evidence for photochemically induced reduction of Fe(III) under seawater conditions. These results are perhaps understandable given the fast reoxidation reactions by hydrogen peroxide and oxygen at seawater pH and the use of membrane filtration to separate soluble and insoluble iron. Reproducible results are obtained in stirred seawater and show a gradual decrease in levels of 0.2 μm filterable iron over time. The light replicates show a slower rate of iron loss from solution than in the dark. The possible implications of these results for iron bioavailability in seawater are discussed.

Temperature Programmed Desorption/Reaction of Ammonia over V2O5/TiO2 De-NOxing Catalysts

Temperature programmed desorption (TPD) of ammonia, temperature programmed surface reaction (TPSR) of adsorbed ammonia with gas phase NO, temperature programmed reaction (TPR), and steady state reaction (SSR) experiments of NH3 + NO under diluted gas conditions in the presence and in the absence of oxygen are performed on a series of V2O5/TiO2 (anatase) catalysts with V2O5 loadings ≤ 3.56% w/w that are comparable to those of commercial De-NOxing catalysts. Both isolated vanadyls and polymeric metavanadate species are observed on the surface of submonolayer vanadia/titania samples, the relative concentration of the latter species increasing with the vanadia content. TPR, TPSR, and SSR experiments performed in the presence and in the absence of oxygen provide new and more direct evidence than previously reported in the literature fora greater reactivity of polymeric metavanadate species as compared to isolated vanadyls and for a faster reduction by NH3 and a faster reoxidation by gaseous oxygen of the polymeric metavanadate groups. This has been related to the presence of more labile oxygen atoms, that is beneficial for the activity but is also detrimental for the selectivity of the SCR reaction. The turnover frequency measured at 500 K under steady state conditions over high-vanadia loading catalysts is six times larger than that of low-vanadia loading catalysts, due to the higher amounts of polyvanadate species. The temperature window of the SCR reaction, corresponding to high NO conversion and complete selectivity to nitrogen, is shifted towards lower temperatures on increasing the vanadia loading and extends towards higher temperatures under steady state conditions and in the presence of oxygen due to a proper oxidation level of the catalyst.

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 μmol photons·m−2·s−1 and became photoinhibited, as indicated by the decrease in Fv/Fm (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound QB, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q a - , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound QB and in QB-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [14C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.

The pathway of electron transfer in NADH:Q oxidoreductase

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.

A critical reevaluation of some assay methods for superoxide dismutase activity

We have compared the direct method of pulse radiolysis to the indirect methods of cytochrome c and nitroblue tetrazolium for assaying the superoxide activity of a compound. We have shown that with pulse radiolysis, where high concentrations of O 2 − are generated, the “turnover” rate constant, k cat, can be determined directly, while with the indirect methods, where relatively low steady state concentrations of O 2 − are formedl, the value of k cat determined by these methods, can be orders of magnitiude lower than that determined directly. The main reason for the lower values obtained with the indirect methods is due to the fast reoxidation of the reduced compound by molecular oxygen. Additional problems which arise with the use of indirect methods for determining superoxide dismutase catalytic activity are discussed.

Comparative study of alumina-supported CuO and CuCr 2O 4 as catalysts for CO oxidation

Alumina-supported CuO and CuCr 2O 4 catalysts of various compositions were prepared and their activity for CO oxidation measured. Fresh, pretreated with CO, and reoxidized catalysts were studied. In general, the activity increased with the CO pretreatment. The extent of the activation depended on the catalyst composition. Thus, copper catalysts were more active than copper chromite at low metal concentrations (<12 wt%), but the opposite was observed at concentrations higher than about 12 wt%. The results on activity behavior, together with TPR and XRD spectra, suggest that the active species are related to copper cations. In the low concentration copper catalysts, the most active surface is generated after CO pretreatment, followed by a fast reoxidation occurring at the first stages of the reaction. In the high concentration copper catalysts, the CO pretreatment produced an induction period as a result of excessive reduction. It is suggested that the role of chromium is to limit the extent of reduction through the formation of the CuCr 2O 4 phase. The presence of this phase also resulted in catalysts less prone to deactivation, as compared to copper on alumina catalysts.

The electrochemistry of proteins and related substances: Part II. Insulin

The electroreduction of insulin in pH 7.4 solution was studied at the hanging mercury drop electrode by cyclic voltammetry and at a mercury pool electrode by controlled potential coulometry. The proposed mechanism involves reduction of an adsorbed monolayer of insulin (maximum coverage of 10 μC cm −2) in a four-electron reaction at about −0.6 V vs. SCE resulting in breakage of two of the three disulfide bonds in the molecule. Fast reoxidation leads to recovery of much of the reduced species. At longer time (ca. 100 s) a steady state is reached where the reductions and reoxidation involve reversible two-electron reactions.