Electrostatic Constituents of the Interaction of Cardiolipin with Site A of Cytochrome c

Abstract

Cytochrome c binds to cardiolipin (CL) on the inner mitochondrial membrane during the initial stages of apoptosis where it oxidizes CL, promoting its release into the cytoplasm where it initiates apoptosis. Previous work has identified interaction sites on cytochrome c involved in the cytochrome c-CL interaction. The contributions of the lysines attributed to site A, the anionic site, are studied here to elucidate the relative importance of each for electrostatic interaction of cytochrome c with CL at pH 8, conditions where site A is dominant. A set of single, double, and quadruple lysine to alanine variants of yeast iso-1-cytochrome c, at sequence positions 72, 73, 86, and 87, show that all contribute to the site A-mediated interaction with CL. All variants experience two sequential structural rearrangements as the lipid to protein ratio (LPR) increases. At a low LPR near 10, all variants undergo a small heme-centered structural change detected by Soret circular dichroism. At higher LPRs ranging from 22 to 34, all variants partially unfold as detected by Trp59 emission. The robustness of the mechanism of interaction to sequential neutralization of the four lysines assigned to site A demonstrates that site A is more extensive than previously supposed. The nature of both structural rearrangements also depends on which lysines constitute site A. The peroxidase activity of cytochrome c in the early stages of apoptosis depends on the nature of structural rearrangement near the heme. Thus, the lysines that comprise site A may have evolved to optimize the peroxidase signaling switch.

Calixarene capture of partially unfolded cytochrome c

“…And the involvement of Pro71 (CH-p interaction with a phenol ring) is also reminiscent of the PAF-sclx 6 complexation (compare Figs 1 and 2C). Calix [6]arene complexation and cytc restructuring is interesting in the context of the alkaline conformational transition, which can involve Lys72, Lys73, or Lys79 on Ω-loop D [8,[10][11][12][13][14]. Apparently, the mobility of this loop facilitates the two different processes.…”

“…Similar to suramin [28], the 'molecular glue' activity of calixarenes can give rise to interesting protein assembly properties [16][17][18][19][20][21][22][23]. For example, phosphonato-calix [6]arene (pclx 6 ) induces cytc dimers [18], while sulfonato-calix [8]arene (sclx 8 ) gives rise to autoregulated tetramer formation [20]. Furthermore, highly porous crystalline architectures have been obtained with cytc and sclx 8 .…”

“…For example, phosphonato-calix [6]arene (pclx 6 ) induces cytc dimers [18], while sulfonato-calix [8]arene (sclx 8 ) gives rise to autoregulated tetramer formation [20]. Furthermore, highly porous crystalline architectures have been obtained with cytc and sclx 8 . In one case a protein-calixarene framework (PDB 6gd9) is mediated exclusively by the calixarene [20].…”

Membrane-tethering of cytochrome c accelerates regulated cell death in yeast

“…This peroxidation weakens the binding of cytochrome c to cardiolipin and facilitates its detachment from the IMM and subsequent release into the cytosol 2,50 . The peroxidase activity of cytochrome c is highly dependent on distinct surface charges facilitating interaction and structural arrangements upon binding to cardiolipin 51,52 . Thus, it is likely that anchoring cytochrome c to the IMM favours its peroxidase activity, leading to a progressive increase of peroxidized cardiolipin that likely sensitizes cells towards cell death induction.…”

Sex differences and gonadal hormone regulation of brain cardiolipin, a key mitochondrial phospholipid

“…The asymmetrical distribution of CL may be a consequence of its ionic charge, its place of synthesis or its interaction with proteins. Indeed, CL establishes non‐covalent bonds with a long list of proteins, including respiratory complexes I, III, IV and V, uncoupling proteins (UCPs), proteins involved in fusion‐fission events, the ADP/ATP carrier, and cytochrome c …”