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

Abstract

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.

Correlation between the potency of flavonoids for cytochrome c reduction and inhibition of cardiolipin‐induced peroxidase activity

“…Moreover, interaction of flavonoids with Lys79 and vicinal basic residues of Cyt c may further potentiate electronic capture by the heme , which could explain the high reducing efficiency of flavonoids like quercetin, kaempferol and cyanidin. Furthermore, a cluster of Lys and His residues at the entrance of the heme crevice constitutes the L site implicated in Cyt c interaction with acidic lipids, with p K a values 6.5–7 , which correlates with the pH‐dependence of the reduction rates by quercetin and kaempferol found in our work.…”

Cytochrome c-promoted cardiolipin oxidation generates singlet molecular oxygen

“…Singlet oxygen was only detected at pH values higher than 6 and was generated at highest yields in the pH range of 8-9. Based on ionization behavior of basic groups at sites A and L, 39,41 these data indicate that singlet oxygen is generated at a condition favouring electrostatic interactions with site A. Further studies are necessary to corroborate this hypothesis.…”

Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

“…His is frequently found interacting with phospholipid headgroups in membrane protein three-dimensional structures, especially in sites for cardiolipin (64). In addition, His often contributes a pH dependence for binding (65,66), becoming cationic at a slightly reduced pH. The more deeply penetrating monotopic proteins also caused a local convex change in bilayer curvature, which was absent for less penetrating species (63).…”