Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, United States.
Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, United States; Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, 15261, United States; Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, 15260, United States.
Free Radic Biol Med. 2021 Jan;162:423-434. doi: 10.1016/j.freeradbiomed.2020.10.321. Epub 2020 Nov 2.
Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of -189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding.
细胞色素球蛋白是一种在哺乳动物组织中广泛表达的保守血红素蛋白，它与一氧化氮（NO）和脂质代谢的信号功能进行电子转移反应。细胞色素球蛋白具有 E7 远端组氨酸（His81），与肌红蛋白和血红蛋白等相关球蛋白不同，它在结合的六配位态和未结合的五配位态之间处于平衡状态。His81 的结合平衡似乎通过两个半胱氨酸（Cys38 和 Cys83）之间的分子内二硫键的存在被别构调节。形成这个二硫键桥调节亚硝酸盐还原酶活性和脂质结合。在此，我们试图通过氰化物结合来探测三价铁状态，阐明定义的巯基氧化态对细胞色素球蛋白血红素小分子结合的影响。氰化物与野生型细胞色素球蛋白的结合动力学揭示，取决于巯基氧化状态，至少存在两种动力学上不同的亚群。用 NEM、谷胱甘肽和氨基酸取代（C38S、C83S 和 H81A）进行的共价巯基修饰实验表明，从完全还原的巯基到单个巯基氧化和分子内二硫键形成的亚群通过调节组氨酸-血红素亲和力和配体结合来确定血红素结合特性。配体结合的氧化还原调节对生理水平的过氧化氢敏感，天然细胞色素球蛋白分子内二硫键的功能中点氧化还原电位为-189±4 mV，这是细胞内氧化还原电位范围内的值。这些结果支持了这样的假设，即细胞色素球蛋白上的 Cys38 和 Cys83 作为敏感的氧化还原传感器，调节细胞色素球蛋白远端血红素口袋的反应性和配体结合。
