Purification and characterization of two-subunit cytochrome aa3 from Bacillus cereus.
Cytochrome c-oxidase type aa3 (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 172 kDa in size; however, only a monomer of 81 kDa was detected when analysed by non-denaturing gel electrophoresis. Denaturing gel electrophoresis analysis of the protein showed the presence of two subunits (51 and 30 kDa). Atomic absorption and visible spectroscopy showed typical aa3 redox centres with haem a iron and copper in a ratio of 22 nmol and 35 ng-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.
Garcia-Horsman
J A
JA
Departamento de Microbiología, Universidad Nacional Autónoma de México, D.F.
Barquera
B
B
Gonzalez-Halphen
D
D
Escamilla
J E
JE
D001407:Bacillus / Q000201:enzymology; D001409:Bacillus cereus / Q000201:enzymology*; D002308:Cardiolipins / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000494:pharmacology; D013057:Spectrum Analysis
D002308:Cardiolipins; D003574:Cytochrome c Group; D005960:Glucosides; D010713:Phosphatidylcholines; D010743:Phospholipids; C040358:dodecyl maltoside; C024990:asolectin; D003576:Electron Transport Complex IV