Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: Comparison to the complex from Escherichia coli

J. F. Kolonay, F. Moshiri, R. B. Gennis, T. M. Kaysser, R. J. Maier

Research output: Contribution to journalComment/debate

Abstract

Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO- reactive component. The complex had a K(m) for ubiquinol-I approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a K(m) value for O2 three times greater than that which we determined for the E. coli bd complex.

Original languageEnglish (US)
Pages (from-to)4177-4181
Number of pages5
JournalJournal of bacteriology
Volume176
Issue number13
DOIs
StatePublished - Jan 1 1994
Externally publishedYes

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Cytochrome d Group
Azotobacter vinelandii
Carbon Monoxide
Cytochromes
Escherichia coli
Agarose Chromatography
Heme
Centrifugation
Sodium Dodecyl Sulfate
Detergents
Polyacrylamide Gel Electrophoresis
Membranes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii : Comparison to the complex from Escherichia coli. / Kolonay, J. F.; Moshiri, F.; Gennis, R. B.; Kaysser, T. M.; Maier, R. J.

In: Journal of bacteriology, Vol. 176, No. 13, 01.01.1994, p. 4177-4181.

Research output: Contribution to journalComment/debate

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AU - Kolonay, J. F.

AU - Moshiri, F.

AU - Gennis, R. B.

AU - Kaysser, T. M.

AU - Maier, R. J.

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N2 - Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO- reactive component. The complex had a K(m) for ubiquinol-I approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a K(m) value for O2 three times greater than that which we determined for the E. coli bd complex.

AB - Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO- reactive component. The complex had a K(m) for ubiquinol-I approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a K(m) value for O2 three times greater than that which we determined for the E. coli bd complex.

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