pH-Dependent interaction of cytochrome c with mitochondrial mimetic membranes: the role of an array of positively charged amino acids.
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 approximately 7.0. 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.
Kawai
Cintia
C
Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, São Paulo CEP 08780-911, Brazil.
Prado
Fernanda M
FM
Nunes
Gabriel L C
GL
Di Mascio
Paolo
P
Carmona-Ribeiro
Ana M
AM
Nantes
Iseli L
IL
D000418:Albumins / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D002308:Cardiolipins / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008246:Lysophospholipids / Q000737:chemistry; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009206:Myocardium / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010767:Phosphorylcholine / Q000737:chemistry; D017785:Photons; D011479:Protamines / Q000737:chemistry; D013053:Spectrophotometry; D055672:Static Electricity
C033518:2-(10-(1-pyrene)decanoyl)phosphatidylcholine; D000418:Albumins; D000596:Amino Acids; D002308:Cardiolipins; D008246:Lysophospholipids; D010714:Phosphatidylethanolamines; D011479:Protamines; D010767:Phosphorylcholine; D006639:Histidine; D002244:Carbon; C038151:1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine; D045304:Cytochromes c; D008239:Lysine