Unraveling cardiolipin-induced conformational change of cytochrome c through H/D exchange mass spectrometry and quartz crystal microbalance

Sin-Cih Sun,Hung-Wei Huang,Yi-Ting Lo,Min-Chieh Chuang,Yuan-Hao Howard Hsu

doi:10.1038/s41598-020-79905-8

Abstract

Cardiolipin (CL), a crucial component in inner mitochondrial membranes, interacts with cytochrome c (cyt c) to form a peroxidase complex for the catalysis of CL oxidation. Such interaction is pivotal to the mitochondrial regulation of apoptosis and is affected by the redox state of cyt c. In the present study, the redox-dependent interaction of cyt c with CL was investigated through amide hydrogen/deuterium exchange coupled with mass spectrometry (HDXMS) and quartz crystal microbalance with dissipation monitoring (QCM-D). Ferrous cyt c exhibited a more compact conformation compared with its ferric form, which was supported by the lower number of deuterons accumulated and the greater amplitude reduction on dissipation. Upon association with CL, ferrous cyt c resulted in a moderate increase in deuteration, whereas the ferric form caused a drastic increase of deuteration, which indicated that CL-bound ferric cyt c formed an extended conformation. These results were consistent with those of the frequency (f) − dissipation (D) experiments, which revealed that ferric cyt c yielded greater values of |ΔD/Δf| within the first minute. Further fragmentation analysis based on HDXMS indicated that the effect of CL binding was considerably different on ferric and ferrous cyt c in the C-helix and the Loop 9–24. In ferric cyt c, CL binding affected Met80 and destabilized His18 interaction with heme, which was not observed with ferrous cyt c. An interaction model was proposed to explain the aforementioned results.

Highlights

Cytochrome c is a heme protein located in the mitochondrial intermembrane space in eukaryotic c ells[1]
A successful synthesis of the CL/DPPC liposome was confirmed using nonyl acridine orange (NAO) labeling, which absorbed with CL (Supplementary Fig. S2)
The results are summarized as follows: (i) CL binding had a different effect on ferric and ferrous cyt c in C-helix and Loop 9–24. (ii) Loop 9–24, containing Fe coordination residue His[18] and the thioether covalent bonding residues Cys[14] and Cys[17], stabilized the relative position of heme and the protein structure. (iii) CL interaction with ferric cyt c affected Met[80] and destabilized His[18] interaction with heme, which was not observed in ferrous cyt c. (iv) The C-helix on ferric cyt c was close to full deuteration after 30 min of deuteration, which indicated a high exposure of this region; such behavior was not observed in ferrous cyt c

Summary

Introduction

Cytochrome c (cyt c) is a heme protein located in the mitochondrial intermembrane space in eukaryotic c ells[1]. Oxidative stress induces peroxidation of CL to inhibit electron transport and alter mitochondrial bioenergetics[5,6,7] Such CL peroxidation changes the integrity of inner mitochondrial membranes and affects the interactions between CL and cyt c3,8–10. The redox state of the heme determines the affinity of cyt c to electron transport chain complex III and complex IV in m itochondria[21,22,23] Ferric cyt c tends to associate with complex III, but ferrous cyt c favors binding to Scientific Reports | (2021) 11:1090. The oxidation state of heme affects the conformation of cyt c and the affinity to the electron transport complexes. The information obtained from these two analytical methods provided an explanation of the interaction mechanism from global and fragmentary perspectives

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Results

Discussion

Conclusion