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

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),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 pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) 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 Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Brazilian research funding agencies FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CNPq, Instituto do Milenio Redoxoma (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - INCT)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Fondo Clemente Estable/ Agencia Nacional de Investigacion e Innovacion,Uruguay

Fondo Clemente Estable/ Agencia Nacional de Investigacion e Innovacion,Uruguay

Howard Hughes Medical Institute

Howard Hughes Medical Institute

International Centre for Genetic Engineering and Biotechnology

International Centre for Genetic Engineering and Biotechnology