Immunological and spectral characterization of partly purified cytochrome oxidase from the cyanobacterium Synechocystis 6714.
Membranes were isolated by French pressure cell extrusion of lysozyme-preincubated cells of the cyanobacterium Synechocystis 6714 after growth in the presence of 0.4 M NaCl for 4 days. These cells showed up to 6-fold respiratory activity (oxygen uptake) when compared to control cells. Separation of plasma and thylakoid membranes revealed that the major part of cytochrome c oxidase was associated with the latter. Immunoblotting of sodium dodecylsulfate polyacrylamide gel electrophorized membranes with antisera raised against subunit I, subunit II, and the holoenzyme of the aa3-type cytochrome oxidase from Paracoccus denitrificans gave specific and complementary cross-reactions at apparent molecular weights of about 25 and 17-18 kDa, respectively. Crude membranes were solubilized also with n-octyl glucoside, and the cytochrome oxidase was separated from the extract by affinity chromatography using immobilized cytochrome c from Saccharomyces cerevisiae. The enzyme was eluted with KCl/octyl glucoside. Dialysed and concentrated enzyme solution, which was free of b- and c-type cytochromes, gave reduced alpha- and gamma-peaks around 603 and 443 nm, respectively. Upon treatment of the sample with carbon monoxide the peaks were found at 593 and 433 nm, respectively. Photodissociation spectra of the CO-complexed enzyme were in full agreement with cytochrome aa3 being a functional cytochrome oxidase in Synechocystis 6714.
Wastyn
M
M
Biophysical Chemistry Group, University of Vienna, Austria.
Achatz
A
A
Trnka
M
M
Peschek
G A
GA
D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D000458:Cyanobacteria / Q000201:enzymology*; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D007118:Immunoassay; D008970:Molecular Weight; D010231:Paracoccus denitrificans / Q000201:enzymology; D012995:Solubility; D013053:Spectrophotometry
D003574:Cytochrome c Group; D005960:Glucosides; C018619:octyl-beta-D-glucoside; D003576:Electron Transport Complex IV