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A study was conducted to investigate that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration. Findings suggest that site L ionization determines the participation of cytochrome c in the respiratory chain and apoptosis.

Fluorescence quenching of lipid-boundpyrene was used to assessthe binding of cytochrome c(cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM)POPC/DOPE/TOCL, with the conditions that it did ordid not contain oxidized phosphatidylcholine molecules, i.e.,1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine(PazePC), or a mixture of two hydroperoxide isomers derivedfrom POPC (POPCOX). The binding isotherms reveal two dissociationconstants, KD1and KD2, representing, respectively, thelow- and high-affinity states of the membrane. These dissociationconstants probably are due to the lipid reorganization promoted bycyt c, as observed in giant unilamellar vesiclesthat contain fluorescent cardiolipin (CL). The presence of PazePC,which has a nonreactive carboxylic group, increased the KD1and KD2values 1.2- and 4.5-fold, respectively. The presence of POPCOXwhich has a reactive peroxide group, decreased the KD1value 1.5-fold, increased the KD2value 10-fold, and significantlyreduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt cincubatedwith liposomes containing POPCox demonstrated a mass increase correspondingto the formation of nonenal adducts as hydrophobic anchors. Electronicabsorption spectroscopy, circular dichroism, and magnetic circulardichroism demonstrated that all of the lipids studied promoted changesin the cyt ccoordination sphere. Therefore,in the presence of CL, the oxidation of zwitterionic lipids also promoteschanges in the cyt cstructure and in the affinityfor lipid bilayers. [ABSTRACT FROM AUTHOR]

Ca 2+ -overload contributes to the oxidation of mitochondrial membrane lipids and associated events such as the permeability transition pore (MPTP) opening. Numerous experimental studies about the Ca 2+ /cardiolipin (CL) interaction are reported in the literature, but there are few studies in conjunction with theoretical approaches based on ab initio calculations. In the present study, the lipid fraction of the inner mitochondrial membrane was modeled as POPC/CL large unilamellar vesicles (LUVs). POPC/CL and, comparatively, POPC, and CL LUVs were challenged by singlet molecular oxygen using the anionic porphyrin TPPS4 as a photosensitizer and by free radicals produced by Fe 2+ -citrate. Calcium ion favored both types of lipid oxidation in a lipid composition-dependent manner. In membranes containing predominantly or exclusively POPC, Ca 2+ increased the oxidation at later reaction times while the oxidation of CL membranes was exacerbated at the early times of reaction. Considering that Ca 2+ interaction affects the lipid structure and packing, density functional theory (DFT) calculations were applied to the Ca 2+ association with totally and partially protonated and deprotonated CL, in the presence of water. The interaction of totally and partially protonated CL head groups with Ca 2+ decreased the intramolecular P-P distance and increased the hydrophobic volume of the acyl chains. Consistently with the theoretically predicted effect of Ca 2+ on CL, in the absence of pro-oxidants, giant unilamellar vesicles (GUVs) challenged by Ca 2+ formed buds and many internal vesicles. Therefore, Ca 2+ induces changes in CL packing and increases the susceptibility of CL to the oxidation promoted by free radicals and excited species., (Copyright © 2020 Miranda, Araujo-Chaves, Kawai, Brito, Dias, Arantes and Nantes-Cardoso.)

Important findings regarding the structure and function of respiratory cytochromes have been made from the study of these hemeproteins associated to liposomes. These studies contributed to the comprehension of the biological role of these proteins in the electron transfer process, the regulatory mechanisms, the energy transduction mechanisms, the protein sites that interact with mitochondrial membranes and the role played by the non-redox subunits present in the protein complexes of the respiratory chain of eukaryotes. In this chapter, the protocols developed to study cytochrome bc (1) activity in liposomes and the binding of cytochrome c to lipid bilayers is presented . The former protocol was developed to study the mechanism of energy transduction related to the topology of the components of bc (1) complex in the mitochondrial membrane. These studies were done with purified cytochrome bc (1) complexes reconstituted into potassium-loaded vesicles. The latter protocol was developed to study the influence of pH, DeltapH, and DeltaPsi on the interaction of cytochrome c with liposomes that mimic the inner mitochondrial membrane.