Reactions of reduced cellobiose oxidase with oxygen. Is cellobiose oxidase primarily an oxidase?
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ABSTRACT:
SUBMITTER: Wilson MT
PROVIDER: S-EPMC1131709 | biostudies-other | 1990 Aug
REPOSITORIES: biostudies-other
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Project description:A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c-oxygen oxidoreductase) in which only cytochrome a(3) is reduced and in combination with CO. The kinetics of CO binding by cytochrome a(3) (2+) in this species is significantly different from that exhibited by cytochrome a(3) (2+) in the fully reduced enzyme. The second-order rate constant for combination was 5x10(4)m(-1).s(-1) and the ;off' constant was 3x10(-2)s(-1). The kinetic difference spectra cytochrome a(3) (2+)-cytochrome a(3) (2+)-CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a(3) (2+) and oxygen in the mixed-valence species was followed in flow-flash experiments and reveals a fast, oxygen-dependent (8x10(7)m(-1).s(-1) at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O(2)]. The fast oxygen-dependent reaction yields as the first product the so-called ;oxygenated' enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a(3) depends on the redox state of its partners, a fact which represents clear evidence for site-site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a(3), is reduced represents clear and unequivocal evidence that this is indeed the O(2)-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.
| S-EPMC1166106 | biostudies-other
Project description:The kinetics of the electron-transfer process which occurs between ferrocytochrome c and partially reduced mammalian cytochrome oxidase were studied by the rapid spectrophotometric techniques of stopped flow and temperature jump. Stopped-flow experiments showed initial very fast extinction changes at 605 nm and at 563 nm, indicating the simultaneous reduction of cytochrome a and oxidation of ferrocytochrome c. During this 'burst' phase, say the first 50 ms after mixing, it was invariably found that more cytochrome c had been oxidized than cytochrome a had been reduced. This discrepancy in electron equivalents may be accounted for by the rapid reduction of another redox site in the enzyme, possibly that associated with the extinction changes observed at 830 nm. During the incubation period in which the partially reduced oxidase was prepared, the rate of reduction of cytochrome a by ferrocytochrome c, at constant reactant concentrations, decreased with time. Temperature-jump experiments showed the presence of two relaxation processes. The faster of the two phases was assigned to the electron-transfer reaction between cytochrome c and cytochrome a. A study of the concentration-dependence of the reciprocal relaxation time for this phase yielded a rate constant of 9 X 10(6)M-1-s-1 for the electron transfer from cytochrome c to cytochrome a, and a value of 8.5 X 10(6)M-1-s-1 for the reverse reaction. The equilibrium constant for the electron-transfer reaction is therefore close to unity. The slower phase has been interpreted as signalling the transfer of electrons between cytochrome a and another redox site within the oxidase molecule.
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Project description:The kinetics of the reaction of fully reduced membrane-bound cytochrome oxidase with O2 obtained in the Soret, alpha and near-i.r. regions were analysed, and the contributions of the three intermediates of the reaction [Clore & Chance (1978) Biochem. J. 173, 799--810] to seven wavelength pairs (430--463, 444--463, 590--630, 608--630, 740--940, 790--940 and 830--940 nm) were determined. The nature of the intermediates is discussed on the basis of the data in the present paper together with data in the literature from optical wavelength scanning, e.p.r., i.r. and magnetic-susceptibility studies.
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Project description:The availability of oxygen (O(2)) in aerated (i.e., water-unsaturated) soils affects the metabolic activities of aerobic and anaerobic soil prokaryotes that degrade plant-derived saccharides. Fluctuating availabilities of O(2) were imposed on agricultural soil slurries supplemented with cellobiose. Slurries were subjected to oxic conditions (48 h), followed by an anoxic period (120 h) and a final oxic period (24 h). Redox potential was stable at 500 mV during oxic periods but decreased rapidly (within 10 h) under anoxic conditions to -330 mV. The consumption of cellobiose occurred without apparent delay at all redox potentials. The metabolic activities of seven previously identified saccharolytic family-level taxa of the investigated soil were measured with newly designed quantitative PCR assays targeting the 16S rRNA. Four taxa responded to the experimental conditions. The amounts of rRNAs of Micrococcaceae and Cellulomonadaceae (Actinobacteria) increased under oxic conditions. In contrast, the RNA contents of Clostridiaceae (cluster I, Firmicutes) and two uncultured family-level-taxa, i.e., "Cellu" and "Sphingo" (both Bacteroidetes) increased under anoxic conditions. That the degradation of cellobiose was independent of the availability of O(2) and that redox potentials decreased in response to anaerobic activities indicated that the degradation of cellobiose was linked to functionally redundant cellobiose-degrading taxa capable of altering redox conditions.
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Project description:The berberine bridge enzyme from the California poppy Eschscholzia californica (EcBBE) catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, that is, the formation of the berberine bridge in the biosynthesis of benzylisoquinoline alkaloids. Interestingly, a large number of BBE-like genes have been identified in plants that lack alkaloid biosynthesis. This finding raised the question of the primordial role of BBE in the plant kingdom, which prompted us to investigate the closest relative of EcBBE in Physcomitrella patens (PpBBE1), the most basal plant harboring a BBE-like gene. Here, we report the biochemical, structural, and in vivo characterization of PpBBE1. Our studies revealed that PpBBE1 is structurally and biochemically very similar to EcBBE. In contrast to EcBBE, we found that PpBBE1 catalyzes the oxidation of the disaccharide cellobiose to the corresponding lactone, that is, PpBBE1 is a cellobiose oxidase. The enzymatic reaction mechanism was characterized by a structure-guided mutagenesis approach that enabled us to assign a catalytic role to amino acid residues in the active site of PpBBE1. In vivo experiments revealed the highest level of PpBBE1 expression in chloronema, the earliest stage of the plant's life cycle, where carbon metabolism is strongly upregulated. It was also shown that the enzyme is secreted to the extracellular space, where it may be involved in later steps of cellulose degradation, thereby allowing the moss to make use of cellulose for energy production. Overall, our results suggest that the primordial role of BBE-like enzymes in plants revolved around primary metabolic reactions in carbohydrate utilization. DATABASE:Structural data are available in the PDB under the accession numbers 6EO4 and 6EO5.
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Project description:Ni- and Co-porphyrin multilayers on reduced graphene oxide (rGO) sheets are reported as novel bifunctional catalysts for the oxygen evolution reaction (OER) and the oxygen reduction reaction (ORR). After binding with organic porphyrin molecules, the catalytically-active Ni2+ and Co2+ ions are periodically constructed onto the rGO surfaces via the layer-by-layer (LBL) assembly technique. The resulting catalysts exhibit good performance towards both OER and ORR, which is achieved with accurate control of the composition and thickness of the multilayer structures. This work highlights the potential for the fabrication of efficient electrocatalysts via molecular design.
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Project description:Reduction of H2O2-oxidized manganese peroxidase (MnP), lignin peroxidase and, to some extent, horseradish peroxidase, was studied in the presence of cellobiose oxidase (CbO) and cellobiose. It was found that the reversion rates for MnP compound II and lignin peroxidase compound II back to native enzymes increased significantly in the presence of CbO and cellobiose. However, the reduction of cytochrome c by CbO plus cellobiose was 40 times faster than the reduction of MnP compound II. Also, the lag phase before reversion to the native states decreased for all three peroxidases in the presence of CbO and cellobiose. Active CbO did not repress formation of compounds I or II of the peroxidases, and Mn2+/veratryl alcohol reduced compound II of the peroxidases much more rapidly than did active CbO. This indicates that, in the presence of Mn2+ or veratryl alcohol, MnP and lignin peroxidase can complete their catalytic cycles and function normally without interference from CbO. Without the presence of peroxidase substrates, active CbO reduced compound II of the above peroxidases.
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Project description:The use of laparoscopic surgery is continuing to increase in colorectal resection and expected reach 80% in the next 10 years. Although laparoscopic (keyhole) or minimally invasive surgery can lead to faster recovery it can also put significant stresses on the patient’s heart and cause fluctuations in blood pressure due to the extreme headdown positioning and abdominal insufflation of carbon dioxide gas.
We have performed several surgical cases under deep neuromuscular block and this has allowed surgery to operate at lower abdominal pressures (from 14 down to 8 mmHg). This put less strain on the heart and allowed higher cardiac outputs.
This study will look at whether deep neuromuscular block is beneficial for patients by
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2. Allowing surgery at lower abdominal insufflation pressures if they have a deep block
3. Reducing patient’s analgesic requirements postoperatively in recovery and at 4 hours
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Project description:1. The results of non-linear optimization studies on the mechanism of reaction of fully reduced cytochrome oxidase with O2 at 176K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at three wavelength pairs (604-630, 608-630 and 830-940 nm) and at three O2 concentrations (60, 200 and 1180 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, good determination of the optimized parameters and a random distribution of residuals is a three-species sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from low-temperature trapping, e.p.r. and magnetic-susceptibility studies, the possible valence states of the metal centres in each of the intermediates are discussed.
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Project description:The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).
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