Among seven homologs of cytochrome b₅₆₁ in a model organism C. elegans, 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 Pichia pastoris 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.

中文翻译：

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秀丽隐杆线虫细胞色素b-2（Cecytb-2）的功能组装到磷脂双层纳米光盘具有增强的铁还原酶活性。

在模型生物秀丽隐杆线虫的七个细胞色素b ₅₆₁同源物中，Cecytb-2被证实在消化器官中表达，被认为是人类Dcytb作为铁还原酶起作用的同源物。Cecytb-2蛋白在毕赤酵母中表达细胞，纯化，并重构为磷脂双层纳米光盘。与去污剂胶束状态相比，在纳米盘环境中重构的Cecytb-2非常稳定，抗坏血酸还原性更高。我们通过分析在厌氧条件下添加铁底物后亚铁血红素的氧化来确认Cecytb-2的铁还原酶活性，在该条件下，观察到的米歇尔-门腾方程对底物浓度具有明显和饱和的依赖性。此外，我们证实通过使用亚硝基-PSAP测定法将铁底物转化为亚铁态。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光盘中得到增强。