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pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

2009; American Chemical Society; Volume: 48; Issue: 35 Linguagem: Inglês

10.1021/bi9006463

1943-295X

Cintia Kawai, Felipe S. Pessoto, Tiago Rodrigues, Kátia Cristina Ugolini Mugnol, Verónica Tórtora, Laura Castro, Vitor Augusto Milicchio, Ivarne L.S. Tersariol, Paolo Di Mascio, Rafael Radí, Ana Maria Carmona‐Ribeiro, Iseli L. Nantes,

ATP Synthase and ATPases Research

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pKa values and site L containing ionizable groups with pKaobs values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, we demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pH-independent binding (microscopic dissociation constant Ksapp2, ∼10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pKa of ∼6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on Ksapp1 was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed Ksapp1 values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site L ionization influences the participation of cytochrome c in the respiratory chain and apoptosis.