pH-dependent Interaction of Cytochrome c with Mitochondrial Mimetic Membranes

Abstract

The interaction of cytochrome c (cyt c) with mitochondrial mimetic vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and heart cardiolipin (PCPECL) was investigated over the 7.4 -6.2 pH range by means of turbidimetry and photon correlation spectroscopy. In the presence of cyt c, the decrease of pH induced an increase in vesicle turbidity and mean diameter resulting from vesicle fusion as determined by a rapid decrease in the excimer/monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidylcholine (PyPC). N-acetylated cyt c and protamine, a positively charged protein, increased vesicle turbidity in a pH-independent manner, whereas albumin did not affect PCPECL vesicle turbidity. pH-dependent turbidity kinetics revealed a role for cyt c-ionizable groups with a pK a(app) of ϳ7.0. The carbethoxylation of these groups by diethylpyrocarbonate prevented cyt c-induced vesicle fusion, although cyt c association to vesicles remained unaffected. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that Lys-22, Lys-27, His-33, and Lys-87 cyt c residues were the main targets for carbethoxylation performed at low pH values (<7.5). In fact, these amino acid residues belong to clusters of positively charged amino acids that lower the pK a . Thus, at low pH, protonation of these invariant and highly conserved amino acid residues produced a second positively charged region opposite to the Lys-72 and Lys-73 region in the cyt c structure. These two opposing sites allowed two vesicles to be brought together by the same cyt c molecule for fusion. Therefore, a novel pH-dependent site associating cyt c to mitochondrial mimetic membranes was established in this study.

The key role played by charge in the interaction of cytochrome c with cardiolipin

“…The models proposed to describe the CL/cyt c interaction identified some regions involved in the protein-liposome interaction [5,6,17,18,26,47]; among others, the Met80-containing 66-92 region, which comprises the cleft formed by the 67-71 and 82-85 residues and a network of positively charged residues (i.e., Lys72, Lys73, Lys86) that may facilitate the insertion of the acyl chain, is particularly noteworthy [5,6,26]. This prompted us to focus our studies on this region, which is considered crucial for the cyt c/CL binding process.…”

Defining the Apoptotic Trigger

“…4c). The first region contains residues Val-20, Lys-22, Gly-24, Lys-25, His-33, Asn-103, and Glu-104, which largely correspond to residues previously proposed to be in the so-called "L" site of CL interaction (46). A second distinct interaction site is composed of Thr-78, Lys-79, Ile-81, and Tyr-48 and corresponds to the previously proposed "A," or anionic, binding site for CL (3).…”

Conformational properties of cardiolipin-bound cytochrome c

“…Cationic residues Lys72, Lys73, Lys86, and Lys87 are thought to participate in electrostatic binding to anionic phospholipids (10,11), with hydrogen bonding between Asn52 and protonated acidic phosholipids further stabilizing CL binding at this site (10,12). Another electrostatic binding site involving residues Lys22, Lys25, His26, Lys27, and His33 has been proposed to play a role at low pH (13). EPR studies have found a partial penetration of cyt c into the lipid bilayer (14), while another study suggested that a hydrophobic acyl chain could also bind into the protein interior (15).…”