Abstract
Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochrome c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300-600 and 200-320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe(3+). The studies also include the effect of P(i), NaCl, and CaCl(2). Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome c with charged interfaces promotes conformational changes in the alpha-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochrome c. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl(2), NaCl, and P(i), in response to the increase of the ionic strength.
Highlights
Cytochrome c is a peripheral membrane protein, and its interaction with negatively charged interfaces is a well known phenomenon [1, 2]
In the present work we examine the different types of cytochrome c/membrane association influenced by the heme iron redox state, membrane charge, and ionic strength
The ferrous cytochrome c magnetic circular dichroism (MCD) spectrum only exhibits a positive band at 418 nm (Fig. 2, top), which accompanies the bathochromic effect observed in the Soret band (Fig. 2, bottom)
Summary
Cytochrome c is a peripheral membrane protein, and its interaction with negatively charged interfaces is a well known phenomenon [1, 2]. The recent demonstration that the apoptosis cascade involves cytochrome c-promoted caspase activation [15, 16] shows that cytochrome c plays a broader role in the cells than electron transport in the respiratory chain In this regard, evidence has been provided for a relationship between apoptosis, the Ca2ϩinduced permeability transition pore [17], and hydrogen peroxide production [18]. In the present work we examine the different types of cytochrome c/membrane association influenced by the heme iron redox state, membrane charge, and ionic strength For this purpose, CD and magnetic circular dichroism (MCD) have been used to detect the conformational alterations in the protein induced by the interfacial microenvironment. EPR was employed to refine the results, providing accurate information about the iron valence, spin state, and change in local symmetry of the iron crystal field around iron
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