Use of lipid vesicles for revealing the potential contribution of cytochrome c in the metabolism of environmental toxicants

Cytochrome c (Cc) is well-known as an electron carrier at mitochondria, but the hemeprotein can also catalyze peroxidase-like reactions. Although it was previously shown that Cc oxidizes aromatic hydrocarbons and heterocyclic compounds, the reported catalytic efficiencies were low. However, Cc interaction with some lipids increases its peroxidase activity. On this basis, we tested the hypothesis that lipid membranes could improve the study of Cc role in the metabolism of environmental toxicants.

The compounds studied were methyl orange (MO), an azo dye model, and the polycyclic aromatic hydrocarbons (PAHs) benzo[b]fluoranthene and benzo[a]pyrene (BaP). The biotransformation assays were carried out at pH 7.0, with Cc from horse heart at 0.01 mg/mL, in the presence of H₂O₂ at 100 microM. Membranes were prepared as small unilamellar vesicles composed of phosphatidylcholine (PC) or mixtures of PC with cardiolipin (CL) at a 4:1 molar ratio. The transformation of MO was monitored by UV-Vis spectrophotometry and the PAHs measured by HPLC.

The results showed that Cc, in the presence of H₂O₂, slowly transformed MO. The presence of the PC vesicles (200 microM) in the reaction media did not significantly affect the transformation kinetics, but the PC/CL vesicles clearly increased the rate of MO decolorization by Cc. In the case of the PAHs, the Cc-mediated oxidation was almost imperceptible (<15%), in spite of the high compounds’ concentrations used in the assays (1 mg/L). Nevertheless, by including CL vesicles in the assay media, the transformation became evident from the decrease observed in the BaP and BbF chromatographic peaks and by the emergence of reaction products peaks in the chromatograms. Indeed, in the presence of these vesicles, BaP oxidation reached 71±2% after 24 h incubations.

In conclusion, lipid membranes can be employed to further investigate the potential participation of Cc in the metabolism of important toxicants.