The Human Cytochrome c Domain-Swapped Dimer Facilitates Tight Regulation of Intrinsic Apoptosis

Abstract

Oxidation of cardiolipin (CL) by cytochrome c (cytc) has been proposed to initiate the intrinsic pathway of apoptosis. Domain-swapped dimer (DSD) conformations of cytc have been reported both by our laboratory and by others. The DSD is an alternate conformer of cytc that could oxygenate CL early in apoptosis. We demonstrate here that the cytc DSD has a set of properties that would provide tighter regulation of the intrinsic pathway. We show that the human DSD is kinetically more stable than horse and yeast DSDs. Circular dichroism data indicate that the DSD has a less asymmetric heme environment, similar to that seen when the monomeric protein binds to CL vesicles at high lipid:protein ratios. The dimer undergoes the alkaline conformational transition near pH 7.0, 2.5 pH units lower than that of the monomer. Data from fluorescence correlation spectroscopy and fluorescence anisotropy suggest that the alkaline transition of the DSD may act as a switch from a high affinity for CL nanodiscs at pH 7.4 to a much lower affinity at pH 8.0. Additionally, the peroxidase activity of the human DSD increases 7-fold compared to that of the monomer at pH 7 and 8, but by 14-fold at pH 6 when mixed Met80/H2O ligation replaces the lysine ligation of the alkaline state. We also present data that indicate that cytc binding shows a cooperative effect as the concentration of cytc is increased. The DSD appears to have evolved into a pH-inducible switch that provides a means to control activation of apoptosis near pH 7.0.

Curvature-Dependent Binding of Cytochrome c to Cardiolipin

“…By contrast, at the highest concentration of iso-1-Cyt c within the vesicle lumen, the perturbation of the Soret CD spectrum is modest. The lowest concentration of iso-1-Cyt c within the lumen of the vesicles is >10-fold higher than the K d reported for Cyt c binding at pH 8 . Therefore, binding to the surface is expected to be complete at all Cyt c concentrations.…”

“…The lowest concentration of iso-1-Cytc within the lumen of the vesicles is >10-fold higher than the K d reported for Cytc binding at pH 8. 15 Therefore, binding to the surface is expected to be complete at all Cytc concentrations. At 5 mM protein concentration, ∼1300 iso-1-Cytc molecules would be contained inside a 100 nm CL vesicle.…”

“…In previous work, we showed that yeast iso-1-Cyt c binds to the convex surface of 100 nm, pure CL vesicles and reported the associated binding parameters for cooperative, one-site, Langmuir-type binding. , These studies were performed at pH 8, conditions selective for binding to the anionic site, A site, − a site which has long been attributed to electrostatic binding via lysines 72 and 73 ,− and possibly lysines 86 and 87 (Figure a) . More recent work suggests that the A site may include other nearby surface lysines (Figure a). , We assigned the two distinct apparent dissociation constants, K d (app), found using Soret circular dichroism (CD) and fluorescence spectroscopy, to a two-step conformational rearrangement on the surface of pure CL vesicles (see Table in the Results section).…”

New Aspects of Cytochrome c: 3D Domain Swapping, Membrane Interaction, Peroxidase Activity, and Met80 Sulfoxide Modification

“…106 Additionally, the human cyt c 3D-DS dimer is kinetically more stable than horse and yeast iso-1-cyt c 3D-DS dimers. 107 The peroxidase activity of human cyt c 3D-DS dimer is 7-fold higher than that of the monomer at pH 7 and 8, but 14-fold higher at pH 6 when mixed Met80/H 2 O ligation replaces the Lys ligation of the alkaline state. 107 As mentioned in the previous section, the proton concentration at the surface of the membrane containing CL is estimated to be higher than that of the bulk.…”

“…107 The peroxidase activity of human cyt c 3D-DS dimer is 7-fold higher than that of the monomer at pH 7 and 8, but 14-fold higher at pH 6 when mixed Met80/H 2 O ligation replaces the Lys ligation of the alkaline state. 107 As mentioned in the previous section, the proton concentration at the surface of the membrane containing CL is estimated to be higher than that of the bulk. 103 These results show that oligomeric 3D-DS cyt c can contribute as a proapoptotic conformer by the increased peroxidase activity.…”