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Purification and characterization of two‐subunit cytochrome aa 3 from Bacillus cereus
Author(s) -
GarciaHorsman J. A.,
Barquera B.,
GonzalezHalphen D.,
Escamilla J. E.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb01840.x
Subject(s) - size exclusion chromatography , cytochrome c oxidase , bacillus cereus , biology , gel electrophoresis , cyanide , cytochrome c , cytochrome , biochemistry , oxidase test , trimer , protein subunit , chromatography , enzyme , dimer , chemistry , bacteria , inorganic chemistry , genetics , gene , mitochondrion , organic chemistry
Summary Cytochrome c ‐oxidase type aa 3 (EC 1.9.3.1.) was purified to homogeneity from vegetative Bacillus cereus by ion‐exchange and hydroxylapatite chromatography in the presence of Triton X–100. Gel filtration analysis suggested a dimeric structure apparently 172kDa in size; however, only a monomer of 81kDa was detected when analysed by non‐denaturing gel electrophoresis. Denaturing gel electrophoresis analysis of the protein showed the presence of two subunits (51 and 30kDa), Atomic absorption and visible spectroscopy showed typical aa 3 redox centres with haem a iron and copper in a ratio of 22 nmol and 35ng‐atom per mg protein, respectively. No haem c was found associated with the purified enzyme in the conditions reported here. Oxidase activity was fully reconstituted by phospholipids in the presence of N, N, N′ N′‐tetramethyl‐ p ‐phenylenediamine or reduced yeast cytochrome c (but not horse cytochrome c) as electron donors. This activity was abolished by cyanide and carbon monoxide.