Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes |
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Authors: | Kapralov Alexandr A Yanamala Naveena Tyurina Yulia Y Castro Laura Samhan-Arias Alejandro Vladimirov Yuri A Maeda Akihiro Weitz Andrew A Peterson Jim Mylnikov Danila Demicheli Verónica Tortora Verónica Klein-Seetharaman Judith Radi Rafael Kagan Valerian E |
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Affiliation: | Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA. |
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Abstract: | Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward 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 H(2)O(2)-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 H(2)O(2)-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. |
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