Surface-Binding to Cardiolipin Nanodomains Triggers Cytochrome c Pro-apoptotic Peroxidase Activity via Localized Dynamics

Abstract

The peroxidation of cardiolipins by reactive oxygen species, which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin (CL), while multidimensional solid-state NMR probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by affecting the dynamics of the cyt c N70-I85 Ω loop, which covers the heme cavity.

Keywords: PUFA; apoptosis; cardiolipin; cytochrome c; lipidomics; membrane oxidation; membrane protein; mitochondrial protein; protein structure and dynamics; solid-state NMR.

Figure 1.. Lipid peroxidase activity of pro-apoptotic cyt c.

(a) X-ray crystal structure of horse heart cyt c (Bushnell et al., 1990), showing the heme (blue spheres), M80 (green sticks), and the 70-85 Ω loop (green highlight). (b) Peroxidase activities of wild-type (WT) and a double mutant (H26N, H33N) cyt c, measured with a fluorescence-based amplex red assay at room temperature. Activities were measured for 0.5 μM cyt c, either without lipids or with TOCL/DOPC LUVs featuring the indicated mol-% of CL at an effective CL/cyt c molar ratio of 4. “Effective CL” accounts for the CL on the outer leaflet of the LUVs. (c) WT cyt c (0.5 μM) peroxidase activity in presence of TOCL/DOPC LUVs with 0, 20, 33, and 50 mol-% CL (effective CL/cyt c ratio = 0, 4, 6.6, 10). Activity values in (b-c) are internally normalized and not comparable to each other. (d) Oxygenated products of TLCL detected by mass spectrometry lipidomics, revealing variants with one to four oxygens added. The amount of product is shown for each mass-to-charge ratio (m/z) and retention time (RT). Control sample lacks cyt c or H₂O₂, while P/L = 1:25 for other samples, with H₂O₂ at 0, 5, and 50 times the cyt c concentration of 1 μM. (e) Quantification of TLCL oxidation by mass spectrometry lipidomics, showing the molar ratio of the sum of oxygenated products to the starting TLCL. (f) CL structure with carbon nomenclature, showing four acyl chains (R₁, R₂, R₃, and R4) connected to the diphosphatidylglycerol through two glycerols. (g) (1) Four identical acyl chains [18:2] of intact TLCL with carbons numbered. (2) Mono-hydroperoxy at C9 of the acyl chain; one predominant oxygenated product of TLCL (m/z 1479, RT 23 min). (3) Oxidative modification of two acyl chains with the addition of one hydroxyl group on each acyl chain at position C9 and C13, respectively (m/z 1479, RT 17 min). All replicates together with their mean and s.d. are plotted in (b)-(e). See also Figure S1 and S2.

Figure 2.. Interaction of cyt c with lipid vesicles of different CL compositions.

(a) Binding of cyt c to TOCL/DOPC vesicles with 12%, 20%, 33%, and 50 mol-% TOCL. The membrane-bound fraction of cyt c is plotted as a function of the effective CL/cyt c molar ratio (bottom axis). The corresponding CL/cyt c area ratio is shown on the top. (b) ³¹P ssNMR spectra of 50 mol-% CL-containing liposome (TOCL and DOPC) with bound cyt c acquired at 10 kHz MAS (top) and under static condition (bottom; orange). The molar L/P ratio is 25:1. The static spectrum of isolated liposomes is shown in black. MAS and static spectra were acquired at a magnetic field of 15.6 T and temperature of 275 K, and 14.1 T and 282 K, respectively. (c) 1D ¹H-¹³C cross polarization (CP) MAS NMR spectrum of U-¹³C,¹⁵N cyt c bound to TLCL/DOPC (1:1) LUVs (L/P = 25:1), at 14.1 T, MAS 8 kHz, and temperature 265 K. Protein and lipid peaks are marked with orange boxes and purple arrows, respectively. (d) SSNMR shows the ¹³C labeled protein (orange boxes) interacts with the membrane and is surrounded by mobile water. 1D ssNMR spectral slices showing ¹³C sites in proximity to mobile acyl chain and water solvent protons, respectively, in absence and presence of 50 ms ¹H-¹H polarization exchange (from 2D ¹H-¹³C spectrum in Figure S3d). 2D spectra were acquired at 8 kHz MAS, 14.1 T, and 265 K at which the bulk water solvent was frozen while lipid-proximal water stayed mobile.

Figure 3.. SSNMR of cyt c bound to TOCL/DOPC LUVs.

(a) Residue-specific NMR chemical shift perturbations (CSP) of cyt c upon binding to the membrane compared to cyt c chemical shifts in solution. Strong (>1.3x average) and medium (>1.1x average) CSP values shown in red and black. The secondary structure of native cyt c (Bushnell et al., 1990) is plotted at top, with heme coordination residues H18 and M80 marked as green stars, and the 70-85 Ω loop in green. (b) 2D ¹³C-¹³C DARR MAS ssNMR spectrum acquired at 252 K, MAS frequency of 10 kHz, and 17.6 T, probing the sedimented LUV-bound protein (1:25 P/L ratio). (c) Strong and medium chemical shift perturbation sites mapped on the cyt c structure in red and salmon, with strong perturbations marked with black labels. The previously proposed “site A” is highlighted in slate blue and labeled in blue. See also Figure S4 and Table S1.

Figure 4.. Residues in 70-85 Ω loop and close to “site A” are affected by local dynamics detected by 2D MAS NMR.

(a) 2D NCA spectra of LUV-bound cyt c (50/50 mol-% TOCL/DOPC; L/P 25:1) acquired at 240 K (mauve), 252 K (green), and 264 K (blue) temperatures. Temperature-dependent changes in peak intensity or position are labeled. (b) Cyt c structure with the affected residues highlighted in cyan. Site A (in slate blue) and heme (in blue) are noted. (c) Spectral regions from 2D ¹³C-¹³C DARR spectra at the same temperatures, showing residues with changing sidechain dynamics. Signals from specific residues labeled in red (Y74 in (1), I81 and I85 in (2) and P76 in (3)) are affected, while others (e.g. F10 in (1), I95 and I75 in (2), and I9 and V11 in (3)) are not. The latter signals are consistent at the different temperatures. (d) Location of residues from (c) highlighted in the cyt c structure in magenta. The spectra were acquired at 15.6 T magnetic field and 10 kHz MAS. See also Figure S5.

Figure 5.. PUFA CL and its impact on cyt c dynamics.

(a) INEPT-based ssNMR 2D spectrum of U-¹³C,¹⁵N-labeled cyt c bound to TLCL/DOPC (1:1) vesicles, P/L ratio 1:25, with protein peaks highlighted in purple labels. The spectrum was acquired at 14.1 T, 285 K, and MAS rate of 10 kHz. (b) INEPT-based 1D ¹³C spectra of TLCL/DOPC (1:1) vesicles with U-¹³C,¹⁵N-labeled cyt c bound, acquired at 285 K and 265 K at 14.1 T and 10 kHz. Cyt c and lipid headgroup signals are marked in the spectra with purple and black arrows, respectively, with unmarked peaks being due to lipid acyl chains. The protein and lipid headgroup signals are missing in the spectrum at 265 K, whereas lipid acyl chain signals are retained. (c) Lipid-dependent perturbations in cyt c backbone (green) and sidechain (pink) comparing binding to TLCL/DOPC (1:1) LUVs, to TOCL/DOPC membrane binding. Site A and heme are marked. Refer also to Figure S5 and S6.

Figure 6.. Schematic model of pro-apoptotic interplay of cyt c and CL in the mitochondrial membrane.

(a) At physiological pH, cyt c binds to negatively charged CL headgroups in the outer leaflet of the mitochondrial inner membrane (MIM) through electrostatic interactions with the so-called “site A” (K72, K73, K86, and K87). Strong association with the membrane involves a cluster of ~6 CL molecules, forming CL-enriched nanodomains. (b) The interaction causes structural and dynamic perturbations of the membrane-facing side of cyt c, surrounding the “site A” lysines (shown orange-red). (c-d) In presence of increased ROS (characteristic of dysfunctional mitochondria), pro-apoptotic lipid peroxidation is catalyzed by the PUFA-CL/cyt c nanocomplex. Although positioned on the surface, cyt-c’s binding to the CL lipids induces dynamics in the 70-85 Ω loop, thereby making the heme cavity more open and accessible. Selective access to the heme cavity is facilitated by the CL nanocluster formation along with the remarkable innate flexibility of the PUFA acyl chains, which are also highly peroxidation-prone.