Identification of b, c, and d cytochromes in the membrane of Vitreoscilla

Georgiou, Christos D.; Webster, D. R.

doi:10.1007/bf00456712

Cited by 16 publications

(9 citation statements)

References 27 publications

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“…Such an approach has been undertaken by Georgiou et al [44], highlighting relationship between cytochromes o from Vitreoscilla sp. C1 and E. coli.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617

Arnaud

Malatesta

Guigliarelli³

et al. 1991

European Journal of Biochemistry

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The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 61 7 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-4791. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDSjPAGE showed the presence of four subunits. The pZ was found to be 4.45 and a M , of 130000 was determined by gel filtration.The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560-nm band for the reduced form in the CI range. The prosthetic group is made of two hemes b, one high-spin ( S = 512, g , = 5.9, gii z 2.0), the other low-spin ( S = 1/2, g, = 2.94, g, = 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one-electron, single redox couple, with Em = 87 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2 . min-' . mg protein-' and commercially available coenzyme (101.74 nmol O2 . min-' . mg protein-'). Exposure of the reduced enzyme to CO induced the collapse of a and bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.Of the terminal enzymes of aerobic respiratory systems, cytochrome-c oxidase of mammalian origin (aa3 type) is the most thoroughly investigated (see reviews in [l -31). This complex transmembrane protein, present in all eukaryotic cells, catalyzes the reduction of molecular oxygen to water in a reaction involving the transfer of four electrons. Coupled to this reaction is a proton pump function which contributes to converting the free energy of O2 reduction by building across the membrane a difference in the proton electrochemical potential. Oxidases of the same aa3-type have been purified from different genera of bacteria. They are far simpler than the enzyme of mammalian origin, being composed of 1 -3 subunits instead of 13 [2, 4, 51. There has, therefore, been a great deal of interest in studying their structures and the mechanisms of the reactions which they catalyze, under far simpler conditions. This is not the only peculiarity of prokaryotic cells, which, in the last few years, provided a number of new insights into electron-transfer mechanisms [6, 71. As far as terminal oxidases are concerned, a great diversity has emerged from investigations of an extending number of bacterial genCorrespondence to M. Denis, Centre d'ocednologie de Marseille,

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“…Such an approach has been undertaken by Georgiou et al [44], highlighting relationship between cytochromes o from Vitreoscilla sp. C1 and E. coli.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617

Arnaud

Malatesta

Guigliarelli³

et al. 1991

European Journal of Biochemistry

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“…Preparation of Membranes and Proteoliposomes-Membranes of both E. coli and Vitreoscilla were obtained by lysozyme treatment of the cells as described previously (32). These were sonicated for 5 min at 25% duty cycle at full power with a Branson Sonifier Cell Disruptor 35 fitted with a microtip to produce the inverted membrane vesicles.…”

Section: Methodsmentioning

confidence: 99%

Vitreoscilla Hemoglobin

Ramandeep

Hwang

Raje

et al. 2001

Journal of Biological Chemistry

136

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The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K d values for the binding of VHb to Vitreoscilla and E. coli cell membranes were ϳ5-6 M, a 4 -8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4-and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

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“…Freshly harvested cells were suspended in an equal volume (w/v) of deionized, sterilized water or 20 mM sodium phosphate buffer (pH 7.2) in a 15-ml plastic bottle and sonicated at 0°C, using repeats of a 30 s burst with a 30 s interval and a 50% duty cycle at power level 7, using a Sonifier 350 (Branson, Danbury, Conn.), for a total of 1 min for each gram of cells. The lysis of Vitreoscilla was performed using the procedure of Georgiou and Webster [13].…”

Section: Cell Lysismentioning

confidence: 99%

Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of Burkholderia and on 2,4-DNT degradation in two-phase bioreactors

Lin

Stark

Webster

2003

Journal of Industrial Microbiology and Biotechnology

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Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions. In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT. The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb. This suggests a direct transfer of oxygen from VHb to the oxygenase. In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.

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Identification of b, c, and d cytochromes in the membrane of Vitreoscilla

Cited by 16 publications

References 27 publications

Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617

Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617

Vitreoscilla Hemoglobin

Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of Burkholderia and on 2,4-DNT degradation in two-phase bioreactors

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