Characterization and application of a thermostable primary transport system: cytochrome-C oxidase from Bacillus stearothermophilus.
Cytochrome-c oxidase from Bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by DEAE-cellulose, hydroxyapatite- and gel-filtration chromatography. The enzyme is a typical cytochrome-aa3-type oxidase which binds carbon monoxide and is sensitive to classical oxidase inhibitors like cyanide and azide. The purified enzyme is composed of three different subunits (57, 37 and 22 kDa). The subunit with intermediate molecular mass contains a covalently attached heme-c moiety. The enzyme appeared to be extremely thermostable (inactivation temperature = 81 degrees C). Highest turnover rates of the reconstituted enzyme were obtained with Saccharomyces cerevisiae cytochrome c or reduced forms of non-physiological electron donors like N,N,N',N'-tetramethyl-p-phenylenediamine and phenazine methosulphate. The reconstituted enzyme can generate a proton-motive force consisting of a high membrane potential and trans-membrane pH gradient. The high electro-motive force of the enzyme (delta p = -180 to -200 mV) indicates that this enzyme functions as a high-capacity electrogenic proton pump. Liposomes containing the purified thermostable and thermoactive cytochrome-c oxidase were fused with membranes from the fermentative bacterium Clostridium acetobutylicum. In the hybrid system a high proton-motive force can be generated upon oxidation of reduced N,N,N',N'-tetramethyl-p-phenylenediamine by the incorporated oxidase which subsequently can be used to drive secondary transport of amino acids. This demonstrates the applicability of the cytochrome-c oxidase to study solute transport in membranes of fermentative bacteria.
De Vrij
W
W
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Heyne
R I
RI
Konings
W N
WN
D002499:Centrifugation, Density Gradient / Q000379:methods; D002845:Chromatography / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D017886:Durapatite; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006882:Hydroxyapatites; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011510:Proteolipids / Q000302:isolation & purification / Q000378:metabolism
D006882:Hydroxyapatites; D046911:Macromolecular Substances; D011510:Proteolipids; C020485:proteoliposomes; D017886:Durapatite; D003576:Electron Transport Complex IV