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D A Webster

Publications and source records attributed to D A Webster.

At least 73 records · Page 4Linked to original sources
PubMed1978

Electron-accepting properties of cytochrome o purified from Vitreoscilla.

The reduction of cytochrome o which contains two hemes/molecule required two electrons/molecule when titrated with dithionite under anaerobic conditions. Two types of spectral transitions occurred during this reduction, first a decrease in absorption bands for the oxidized protein at 540 and 405 nm and then a shift in the absorption maxima to 555 and 425 nm, respectively. Each of these two transitions required approximately one electron, evidence that the two hemes reduced separately. Preliminary estimates of the midpoint potentials of the two hemes enabled the selection of two suitable oxidation-reduction dyes, toluylene blue (E'o = +0.115 V) and indigo carmine (E'o = -0.125 V) which were used to estimate more accurately the midpoint potentials of the high and low potential hemes, respectively, using equilibrium photochemical titrations with EDTA and flavin mononucleotide. The midpoint potentials of the two hemes of cytochrome o determined by this technique were +0.118 and -0.122 V.

Anaerobiosis↗
PubMed1978

Physiological role of oxygenated cytochrome o: observations on whole-cell suspensions of Vitreoscilla.

The form of cytochrome o that predominates in Vitreoscilla cells having various levels of respiratory activity was studied by using untreated, frozen-thawed, and starved cells, which had respiratory rates decreasing in the order given. Direct spectral observation revealed that the oxygenated form of cytochrome o predominated during the aerobic steady-state oxidation of endogenous substrate or exogenous glutamate in untreated and frozen-thawed cells and was replaced by the reduced form when the cell suspensions became anaerobic. The respiratory rates, estimated inversely from the time of duration of the steady state, were correlated to the rates of oxygen consumption for the various cells. Oxidized cytochrome o predominated in aerobic starved cells. These results indicate the involvement of three forms of cytochrome o--oxidized, reduced, and oxygenated--in the catalytic and cyclic change of this cytochrome. The oxygenated form also appeared after the addition of hydrogen peroxide to the cells, but only the oxidized form appeared when ethyl hydrogen peroxide was added. The appearance of the oxygenated form with the addition of hydrogen peroxide was probably due to the reaction of the reduced cytochrome with the oxygen that had evolved by the action of catalase present in the cells.

Aerobiosis↗
PubMed1977

Oxygenated Cytochrome o. An active intermediate observed in whole cells of Vitreoscilla.

The oxygenerated form of cytochrome o can be readily observed in whole cell suspensions of Vitreoscilla because it is the predominant steady state form of the cytochrome in respiring cells. When oxygen is introduced to an anaerobic suspension of cells, the steady state is established immediately and absorption maxima are observed at 578, 545, and 425 nm in difference spectra using ammonium persulfate-oxidized cells as reference. The oxygenerated form of cytochrome o is identified as the predominant steady state species during turnover of the cytochrome in vivo from the close coincidence of these spectra with those obtained using purified cytochrome. When the dissolved oxygen is consumed, the spectrum of the oxygenated form is replaced by the spectrum of the reduced form. Glutamate increased the respiration of these cells and decreased the duration of the steady state. Spectra of the oxygenated form of cytochrome o could also be obtained by using starved cells as reference, i.e. (untreated cells) - (starved cells) difference spectra. These results implicate the oxygenated form of cytochrome o as an active intermediate in terminal electron transport of these cells. In the presence of 1 mM cyanide, which inhibits the respiration of Vitreoscilla, the duration of the steady state was increased and the steady state spectrum was altered, showing that turnover of the cytochrome was inhibitied and formation of the oxygenated form wsa decreased. All these results suggest that the cyclic changes of cytochrome o in terminal respiration involve the oxidized, reduced, and oxygenated forms.

Bacteria↗
PubMed1975

The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla.

The formation of hydrogen peroxide during the oxidation of NADH by purified preparations of cytochrome o has been demonstrated by employing three independent methods: polarographic, colorimetric, and fluorometric. The first two methods were used to assay for the accumulation of hydrogen peroxide and showed that hydrogen peroxide did accumulate as a product, but only about 30% of the oxygen consumed or 15 to 20% of the NADH oxidized was recoverable as hydrogen peroxide. This lack of 1:1 stoichiometry was not due to residual catalase activity in these preparations which could be eliminated by freeze-thawing. Thus, hydrogen peroxide may not be the sole or primary product of the NADH-cytochrome o oxidase reaction. The fluorometric assay could be coupled directly to the NADH-cytochrome o oxidase reaction in one medium, and this method showed that hydrogen peroxide was generated continuously from the beginning of the reaction in a 1:1 stoichiometry, hydrogen peroxide generated to NADH oxidized. This result suggests that hydrogen peroxide is an intermediate that can be trapped efficiently under the conditions of the fluorometric assay, whereas under the conditions of the first two assays most of the hydrogen peroxide generated undergoes further reaction. Exogenously added FAD or FMN increased the percentage of hydrogen peroxide that accumulated in the NADHcytochrome o oxidase reaction. Flavin is believed to act on the reductase side of cytochrome o so the increased percentage of hydrogen peroxide is not likely to result from the direct reaction of reduced flavin with oxygen.

Bacteria↗
PubMed1966

Respiratory chain of colorless algae. II. Cyanophyta.

Whole cell difference spectra of the blue-green algae, Saprospira grandis, Leucothrix mucor, and Vitreoscilla sp. have one, or at the most 2, broad alpha-bands near 560 mmu. At -190 degrees these bands split to give 4 peaks in the alpha-region for b and c-type cytochromes, but no alpha-band for a-type cytochromes is visible. The NADH oxidase activity of these organisms was shown to be associated with particulate fractions of cell homogenates. The response of this activity to inhibitors differed from the responses of the NADH oxidase activities of particulate preparations from the green algae and higher plants to the same inhibitors, but is more typical of certain bacteria. No cytochrome oxidase activity was present in these preparations. The respiration of Saprospira and Vitreoscilla can be light-reversibly inhibited by CO, and all 3 organisms have a CO-binding pigment whose CO complex absorbs near 570, 535, and 417 mmu. The action spectrum for the light reversal of CO-inhibited Vitreoscilla respiration shows maxima at 568, 534, and 416 mmu. The results suggest that the terminal oxidase in these blue-greens is an o-type cytochrome.

Eukaryota↗
PubMedDate not supplied

Bacterial inhibition by electrical activation of percutaneous silver implants.

Percutaneous silver wire implants were looped through the dorsal skin of rats and inoculated with Staphylococcus aureus to test the effect on bacteria in the tract. The silver was activated with four brief daily applications of anodic microcurrent. Contralateral 316L stainless steel implants, identically inoculated, served as controls. Cultures from the silver tracts showed a marked reduction or elimination of bacteria, which persisted for the 3-week study period. In tracts with colonization established for 1 week, subsequent electrical activation of the silver also suppressed the bacteria. Inflammatory reactions at 3 weeks were mild at both the silver and stainless implants and no giant cells or toxicity were seen. This suggests that electrically activated silver may be useful in preventing or treating infection at percutaneous devices.

Administration, Topical↗
PubMedDate not supplied

Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E. coli: direct evidence that VHb enhances recombinant protein production.

Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (VHb) typically produce more protein than unengineered cells, and it has generally been assumed that VHb is responsible for this effect. Here, using matched strains of E. coli that bear a recombinant alpha-amylase gene (MK57) or the alpha-amylase gene and vgb (MK79), we provide evidence supporting this assumption. Sodium nitrite (which is known to inhibit heme proteins) was tested over a range of concentrations regarding effects on growth, alpha-amylase production, respiration, and VHb function in MK57 and MK79. Nitrite concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains, while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57. This suggests that these concentrations inhibit VHb while having a much smaller effect on cytochrome oxidase. Direct measurements of VHb showed, in fact, that the same nitrite concentrations greatly decreased the levels of active (ferrous) and, to a somewhat lesser extent, total (ferrous plus ferric) VHb in MK79. Finally, these same nitrite concentrations reversed the advantage regarding alpha-amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb with the increase in alpha-amylase production.

Escherichia coli↗
PubMedDate not supplied

Effects of culture conditions on enhancement of 2,4-dinitrotoluene degradation by Burkholderia engineered with the Vitreoscilla hemoglobin gene.

Growth and degradation of 2,4-dinitrotoluene (2,4-DNT) were compared in liquid cultures in shake flasks for Burkholderia sp. strain DNT and strain DNT engineered to produce Vitreoscilla (bacterial) hemoglobin (strain YV1). Parameters varied included aeration rate, initial 2,4-DNT concentration (50 and 200 ppm), and concentration and type of cosubstrate (yeast extract, succinate, casamino acids, and tryptic soy broth). 2,4-DNT degradation increased with increasing cosubstrate concentration and was greater for strain YV1 than strain DNT under most conditions tested; the greatest advantages of YV1 (up to 3.5-fold) occurred under limited aeration. A third strain (YV1m), derived from YV1 by repeated growth on 2,4-DNT-containing medium, demonstrated increased 2,4-DNT degradation (up to 1.3-fold compared to YV1) at 200 ppm 2,4-DNT. The growth profiles of the three strains with respect to each other were in general similar to those of the degradation patterns of 2,4-DNT.

Burkholderia↗