title: Phosphomimetic substitution of cytochrome C tyrosine 48 decreases respiration and binding to cardiolipin and abolishes ability to trigger downstream caspase activation.
abstract: Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.
authors.affiliation: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Petr
authors.initials: P
authors.lastname: Pecina
authors.forename: Grigory G
authors.initials: GG
authors.lastname: Borisenko
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Belikova
authors.forename: Yulia Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: Alena
authors.initials: A
authors.lastname: Pecinova
authors.forename: Icksoo
authors.initials: I
authors.lastname: Lee
authors.forename: Alejandro K
authors.initials: AK
authors.lastname: Samhan-Arias
authors.forename: Karin
authors.initials: K
authors.lastname: Przyklenk
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
authors.forename: Maik
authors.initials: M
authors.lastname: Hüttemann
mesh_terms: D000818:Animals; D017209:Apoptosis; D002308:Cardiolipins / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D019069:Cell Respiration; D045304:Cytochromes c / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010755:Organophosphates / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D002308:Cardiolipins; D010755:Organophosphates; D014443:Tyrosine; D045304:Cytochromes c; D020169:Caspases