Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes

Abstract

Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective towards peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues – via the generation of tyrosyl radicals (Tyr•) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H2O2-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H2O2-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.

Cardiolipin Interactions with Proteins

“…This represents a potentially rich source of structural features for analysis and prediction of CL-binding sites. CL-protein docking predictions have been used successfully in the structural analysis of CL interactions previously (9,(113)(114)(115)(116) and have been useful in shedding light on the mechanisms governing such interactions. We therefore docked TLCL, the most abundant CL species in mitochondria, to all structures after removing all bound ligands in them, and analyzed the results (see Materials and Methods section).…”

NO2−-mediated nitrosylation of ferrous microperoxidase-11

“…The fifth coordination ligand of the heme-Fe atom of cyt c is invariantly His, whereas the sixth coordination ligand exhibits high variability, being Met in horse heart cyt c [1][2][3][4][5][6][7]. The heme-Fe atom of hexa-coordinated native horse heart cyt c is essentially unreactive (or very low reactive), whereas the heme-Fe atom of penta-coordinated cyt c derivatives (i.e., carboxymethylated cyt c (CM-cyt c), cardiolipinbound CM-cyt c (CL-CM-cyt c), and cardiolipin-bound cyt c (CL-cyt c)) displays myoglobin-like spectroscopic and functional properties [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”

“…1), the sixth heme-Fe coordination ligand being the O atom of a water molecule in the ferric form. Therefore, MPs are heme-model compounds of choice to highlight the modulatory role of the protein matrix and of the heme-Fe atom axial ligation on the spectroscopic, ligand binding, and (pseudo-)enzymatic properties of horse heart cyt c. In particular, MPs represent heme-model compounds of highly reactive penta-coordinated CM-cyt c, CL-CM-cyt c, and CL-cyt c derivatives, the last being involved in apoptosis initiation [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22].…”

Dual Function of Mitochondrial Nm23-H4 Protein in Phosphotransfer and Intermembrane Lipid Transfer

“…However, CL can also appear in the OM with numerous biological consequences. It can exchange with mitochondrion-associated endoplasmic reticulum for further CL remodeling or interfere with mitochondrial fusion (16,17), and it can also play a role in apoptotic signaling (by recruiting pro-apoptotic proteins (18 -22)) or mitophagy (3,(23)(24)(25). Despite this importance of mitochondrial CL trafficking, the molecular mechanisms involved and their regulation are poorly defined.…”