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.

Figure 1.

Eyring plots for dissociation of human WT (green), equine WT (orange) and yeast WT* (K72A, C102S mutations) (red) DSD dimers. The solid curve is a fit to a form of the Eyring equation that accounts for ${∆\mathit{C}}_{\mathit{p}}^{‡}$ (Eq. 1). The reference temperature (T_o) was set to 310.15 K (37 °C) for all fits. The equine and yeast data are from previously published results.

Figure 2.

Plot of A_695corr versus pH for the alkaline transition for monomeric (closed circles) and DSD (open circles) cytc. Data were collected at room temperature (22 ± 3 °C) in 100 mM NaCl solution with a protein concentration of 100 μM heme (100 μM monomer or 50 μM dimer). Solids lines are fits to eq 2 in materials and methods.

Figure 3.

Plots of % bound monomeric (left) and DSD (right) Zncytc versus CL and DMPC nanodisc concentration (logarithmic scale) as determined by FCS and fluorescence anisotropy. Data points shown are from the FCS experiments. The fits of FCS (solid lines) and anisotropy (dashed lines) data sets to eq 8 are shown. Experimental data were corrected to 25 °C to determine binding and performed in 20 mM TES at the indicated pH and salt concentration.

Figure 4.

Soret CD spectra of human monomer (solid) and DSD (dashed) cytc at a concentration of 10 μM heme (10 μM monomer or 5 μM DSD). Spectra were acquired in 20 mM TES buffer, 0.1 mM EDTA (pH 8.0) at 25 °C.

Figure 5.

Human cytc Soret CD signal as a function of concentration of cardiolipin ND, [ND]. Data points at each [ND] follow the change in amplitude of the peak of the Soret CD signal. For the monomer the changes at 418-nm and for the dimer the changes at 410-nm are plotted versus [ND]. The experimental concentration of cytc was 10 μM based on the heme absorbance (i.e., 10 μM monomer or 5 μM DSD). Experiments were performed in 20 mM TES buffer and 0.1 mM EDTA (pH 8) at 25 °C. Solid curves are fits to eq S14 in the SI.

Figure 6.

k_cat vs pH for monomeric and DSD cytc at 25 °C. Error bars are the standard deviations from three independent experiments. Human data in the absence of CL NDs are from Nold et al.