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.

Organotelluranes exhibit potent antioxidant properties as well as the ability to react with protein thiol groups and, thereby, they are good models to study the mechanism of the mitochondrial permeability transition (MPT). We evaluated the effects of the concentration of organotelluranes, namely RT-03 and RT-04, on rat liver mitochondria. At the concentration range of 0.25-1.0 microM, organotelluranes did not cause any mitochondrial dysfunction. At the concentration range of 5-10 microM, RT-03 and RT-04 caused the Ca2+-dependent opening of the (MPT) pore, regulated by Cyclosporin A. At the concentration range of 15-30 microM the swelling was not inhibited by Cyclosporin A and in the absence of Ca2+, a significant decrease of respiratory control ratio was observed due to concomitant phosphorylation impairment and uncoupling, transmembrane potential disruption, depletion of mitochondrial reduced thiol groups, and alterations in the bilayer fluidity. Above 100 microM, the organotelluranes caused complete inhibition of respiratory chain. Over the whole studied concentration range, RT-03 and RT-04 did not induce mitochondrial oxidative stress assessed by using the reactive oxygen and nitrogen species indicator 2',7'-dichlorodihydrofluorescein diacetate. Further, the organotelluranes also exhibited protective effect against t-butyl hydroperoxide-induced oxidative stress as well as against Fe2+/citrate-induced peroxidation of mitochondrial membranes and PCPECL liposomes. These results point out that MPT pore opening can involve damage exclusively to mitochondrial membrane proteins. The exclusive antioxidant activity observed at nanomolar range is also an interesting new finding described in this work.

In this paper, we present a study about the influence of the porphyrin metal center and meso ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca2+. Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle.