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 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, approximately 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pKa of approximately 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.
Kawai
Cintia
C
Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes (UMC), Mogi das Cruzes, São Paulo, Brazil.
Pessoto
Felipe S
FS
Rodrigues
Tiago
T
Mugnol
Katia C U
KC
Tórtora
Veronica
V
Castro
Laura
L
Milícchio
Vitor A
VA
Tersariol
Ivarne L S
IL
Di Mascio
Paolo
P
Radi
Rafael
R
Carmona-Ribeiro
Ana Maria
AM
Nantes
Iseli L
IL
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D045304:Cytochromes c / Q000378:metabolism*; D004579:Electron Transport / Q000502:physiology*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014413:Tuna
D045304:Cytochromes c; D008239:Lysine