Controlled proteolysis by subtilisin as a probe for cyanide-induced conformational changes in Pseudomonas cytochrome oxidase.
Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c-551:oxidoreductase; EC 1.9.3.2), digested with subtilisin in the absence and presence of KCN, produces discrete, high molecular weight fragments. The presence of KCN alters the rate of this fragmentation but does not change the nature of the fragments. When digested in the absence of KCN, the oxidase gives a major product (A) which is enzymatically active and has an apparent Mr = 58,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of KCN, the major product (B) has Mr = 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but with gel permeation high performance liquid chromatography it has an apparent Mr = 92,000. This implies that product B is dimeric, as is the parent enzyme which has Mr = 110,000 by high performance liquid chromatography. Absorption spectra of product B, isolated by gel filtration, show that it contains only the heme d1 moiety. The digestion time course indicates that the rate at which several minor products are formed is also dependent on the absence or presence of KCN. These observations suggest that the binding of KCN to the heme centers induces a conformational change in the enzyme so that the heme c-containing portion of the protein, which is at one end of the intact enzyme, can be removed without disrupting the integrity of the heme d1-containing portion.
D002851:Chromatography, High Pressure Liquid; D003486:Cyanides / Q000494:pharmacology*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D008970:Molecular Weight; D011190:Potassium Cyanide / Q000494:pharmacology*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013053:Spectrophotometry; D013381:Subtilisins / Q000378:metabolism*
D003486:Cyanides; D003576:Electron Transport Complex IV; D013381:Subtilisins; D011190:Potassium Cyanide