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
Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.
Biochim Biophys Acta. 2011 Sep;1808(9):2147-55. doi: 10.1016/j.bbamem.2011.04.009. Epub 2011 Apr 29.
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.
细胞色素c（cyt c）/心磷脂（CL）过氧化物酶复合物对多不饱和CL过氧化具有选择性，其形成是线粒体膜通透性改变的先决条件。酪氨酸残基——通过生成酪氨酸自由基（Tyr）——可能是导致CL过氧化的过氧化物酶循环的反应中间体。我们使用了马心脏cyt c的突变体，其中四个Tyr残基中的每一个都被苯丙氨酸取代，并评估了它们对过氧化物酶催化的贡献。Tyr67Phe突变与cyt c/CL复合物加氧酶功能的部分丧失以及电子顺磁共振（EPR）光谱中H2O2诱导的Tyr自由基的最低浓度相关。我们的质谱实验直接证明了Tyr67Phe突变体产生的CL氢过氧化物（CL-OOH）减少。同样地，酚类底物Amplex Red的氧化在Tyr67Phe突变体中比在其他三个突变体中受到的影响更大。Tyr67Phe突变体对H2O2诱导的寡聚化具有高度抗性。Tyr荧光测量、异核磁共振（NMR）和计算机模拟表明，Tyr67紧邻卟啉环血红素铁以及两个轴向血红素铁配体残基之一Met80。因此，高度保守的Tyr67可能是cyt c/CL过氧化物酶复合物加氧酶半反应中的电子供体（自由基受体）。
