Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Biochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, Egypt.
Biomolecules. 2021 Jan 13;11(1):96. doi: 10.3390/biom11010096.
Among seven homologs of cytochrome in a model organism , Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.
在模式生物中的七种细胞色素 c 同源物中，Cecytb-2 被证实表达于消化器官，被认为是与人 Dcytb 同源的铁还原酶。Cecytb-2 蛋白在 细胞中表达、纯化，并重组到磷脂双层纳米盘中。在纳米盘环境中重组的 Cecytb-2 极其稳定，与去污剂胶束状态相比，其与抗坏血酸的还原能力更强。我们通过分析在厌氧条件下添加铁底物时亚铁血红素的氧化，证实了 Cecytb-2 的铁还原酶活性，观察到明显的、遵循米氏方程的底物浓度的饱和依赖性。此外，我们证实了使用亚硝基 PSAP 测定法，铁底物被转化为亚铁状态。重要的是，我们观察到 Cecytb-2 的铁还原酶活性在磷脂双层纳米盘中增强。
