The key role played by charge in the interaction of cytochrome c with cardiolipin

Abstract

mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys → Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein.Abstract Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c-CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such Electronic supplementary material The online version of this article

Studying the TRAF2 binding to model membranes: The role of subunits dissociation

“…LUVs were prepared as previously described and laurdan incorporated before vesicles formation (final lipids concentration 1 mM; final laurdan concentration 50 μM).…”

New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains

“…LUVs were prepared as previously described [24] and laurdan incorporated before vesicles formation (final lipids concentration 1 mM; final laurdan concentration 50 µM). GUVs were prepared by the electroformation method developed by Angelova and Dimitrov [25–26] in a special Teflon temperature-controlled chamber, designed by Bagatolli and Gratton [27].…”

An optimal surface concentration of pure cardiolipin deposited onto glassy carbon electrode promoting the direct electron transfer of cytochrome-c

“…[3,4] Its specific function arises from its unique dimeric structure containing four unsaturated alkyl chains and an overall net negative charge at neutral pH ( Figure 1). [5] In addition to its key role for maintaining optimal activity of numerous mitochondrial processes such as electron transfer in the respiratory chain, cardiolipin is also involved in the initiation of the cell apoptosis machinery in particular by forming a peroxidase complex with cytochrome-c. [2,4,[6][7][8][9][10][11][12][13][14] Indeed a direct relationship between cardiolipin loss and cytochrome-c release into the cytoplasm was identified as an initial step in the pathway to apoptosis. [2] The specific and selective interaction between cardiolipin and cytochrome-c is not well understood and has been assigned to a combination of electrostatic and hydrophobic effects, hydrogen bonding and/or the formation of a cardiolipin/cytochrome-c complex.…”

“…[2] The specific and selective interaction between cardiolipin and cytochrome-c is not well understood and has been assigned to a combination of electrostatic and hydrophobic effects, hydrogen bonding and/or the formation of a cardiolipin/cytochrome-c complex. [2,[6][7][8][9][10][11][12][15][16][17][18] The electrochemical behavior of redox proteins such as cytochrome-c, a water-soluble haemoprotein involved in the respiratory chain of mitochondria, has been extensively reported since 1977. [19][20][21][22] This model redox protein has often been studied at lipid-modified electrodes because the detection of its electroactivity is seldom possible at bare electrode surfaces even with large scanning potential range.…”

“…after three cyclic voltammograms. Thus, the immobilization of cytochrome-c onto the modified glassy carbon electrode surface is assigned to specific and selective interactions between cytochrome-c and cardiolipin as documented in the literature [2,[6][7][8][9]15]. The electron transfer rate constant k ET of cytochrome-c immobilized on cardiolipin-modified glassy carbon electrode has been estimated through numerical simulations[51, 52] of the cyclic voltammograms and amounts to 1.5 ± 0.5 s -1 .…”