Wan Junmei, Morse Paul T, Zurek Matthew P, Turner Alice A, Vaishnav Asmita, Salomon Arthur R, Edwards Brian F P, Arroum Tasnim, Hüttemann Maik
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, USA.
Department of Biochemistry, Microbiology and Immunology, Wayne State University, Detroit, MI 48201, USA.
Cells. 2025 Jun 21;14(13):951. doi: 10.3390/cells14130951.
Cytochrome (Cyt) is a multifunctional protein, essential for respiration and intrinsic apoptosis. Post-translational modifications of Cyt have been linked to physiological and pathophysiologic conditions, including cancer. Cyt tyrosine 67 (Y67) is a conserved residue that is important to the structure and function of Cyt We here report the phosphorylation of Y67 of Cyt purified from bovine heart mapped by mass spectrometry. We characterized the functional effects of Y67 Cyt modification using in vitro and cell culture models. Y67 was mutated to the phosphomimetic glutamate (Y67E) and to phenylalanyl (Y67F) as a control. The phosphomimetic Y67E Cyt inhibited cytochrome oxidase (COX) activity, redirecting energy metabolism toward glycolysis, and decreased the pro-apoptotic capabilities of Cyt. The phosphomimetic Y67E Cyt showed a significantly impaired rate of superoxide scavenging and a reduced rate of oxidation by hydrogen peroxide suggesting a lower ability to transfer electrons and scavenge reactive oxygen species (ROS). Phosphomimetic Y67E replacement led to an almost complete loss of cardiolipin peroxidase activity, pointing to a central role of Y67 for this catalytic function of Cyt. In intact cells, phosphomimetic replacement leads to a reduction in cell respiration, mitochondrial membrane potential, and ROS levels. We propose that Y67 phosphorylation is cardioprotective and promotes cell survival.
细胞色素（Cyt）是一种多功能蛋白质，对呼吸作用和内源性细胞凋亡至关重要。Cyt的翻译后修饰与包括癌症在内的生理和病理生理状况有关。Cyt酪氨酸67（Y67）是一个保守残基，对Cyt的结构和功能很重要。我们在此报告通过质谱法对从牛心脏纯化的Cyt的Y67磷酸化进行的定位。我们使用体外和细胞培养模型表征了Y67 Cyt修饰的功能效应。将Y67突变为拟磷酸化谷氨酸（Y67E）并突变为苯丙氨酸（Y67F）作为对照。拟磷酸化的Y67E Cyt抑制细胞色素氧化酶（COX）活性，使能量代谢转向糖酵解，并降低Cyt的促凋亡能力。拟磷酸化的Y67E Cyt显示超氧化物清除速率明显受损，过氧化氢氧化速率降低，表明其转移电子和清除活性氧（ROS）的能力较低。拟磷酸化的Y67E替代导致心磷脂过氧化物酶活性几乎完全丧失，表明Y67在Cyt的这种催化功能中起核心作用。在完整细胞中，拟磷酸化替代导致细胞呼吸、线粒体膜电位和ROS水平降低。我们提出Y67磷酸化具有心脏保护作用并促进细胞存活。
