title: Chronic treatment with azide in situ leads to an irreversible loss of cytochrome c oxidase activity via holoenzyme dissociation.
abstract: Chronic treatment of cultured cells with very low levels of azide (I(50)<10 microm) leads to slow (t(12) = 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azide-mediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl(2), Cu-His) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either Cu(A) or Cu(B). In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa(3) content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties.
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Scot C
authors.initials: SC
authors.lastname: Leary
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Hill
authors.forename: Carrie N
authors.initials: CN
authors.lastname: Lyons
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Carlson
authors.forename: Denise
authors.initials: D
authors.lastname: Michaud
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Kraft
authors.forename: Kenton
authors.initials: K
authors.lastname: Ko
authors.forename: D Moira
authors.initials: DM
authors.lastname: Glerum
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Moyes
mesh_terms: D001386:Azides / Q000494:pharmacology*; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D001386:Azides; D012333:RNA, Messenger; D002374:Catalase; D013482:Superoxide Dismutase; D003576:Electron Transport Complex IV