Facultad de Ciencias Biológicas, Centro de Bioinformática y Biología Integrativa, Universidad Andrés Bello, Santiago, Chile.
Protein J. 2014 Jun;33(3):211-20. doi: 10.1007/s10930-014-9552-3.
The recombinant Chlorobium tepidum ferritin (rCtFtn) is able to oxidize iron using ferroxidase activity but its ferroxidase activity is intermediate between the H-chain human ferritin and the L-chain human ferritin. The rCtFtn has an unusual C-terminal region composed of 12 histidine residues, as well as aspartate and glutamate residues. These residues act as potential metal ion ligands, and the rCtFtn homology model predicts that this region projects inside the protein cage. The rCtFtn also lacks a conserved Tyr residue in position 19. In order to know if those differences are responsible for the altered ferroxidase properties of rCtFtn, we introduced by site-directed mutagenesis a stop codon at position 166 and a Tyr residue replaced Ala19 in the gene of rCtFtn (rCtFtn 166). The rCtFtn166 keeps the canonical sequence considered important for the activity of this family of proteins. Therefore, we expected that rCtFtn 166 would possess similar properties to those described for this protein family. The rCtFtn 166 is able to bind, oxidize and store iron; and its activity is inhibit by Zn(II) as was described for other ferritins. However, the rCtFtn 166 possesses a decrease ferroxidase activity and protein stability compared with the wild type rCtFtn. The analysis of the Ala19Tyr rCtFtn shows that this change does not affect the kinetic of iron oxidation. Therefore, these results indicate that the C-terminal regions have an important role in the activity of the ferroxidase center and the stability of rCtFtn.
重组嗜热菌铁蛋白（rCtFtn）具有通过亚铁氧化酶活性氧化铁的能力，但它的亚铁氧化酶活性介于 H 链人铁蛋白和 L 链人铁蛋白之间。rCtFtn 具有一个不寻常的 C 末端区域，由 12 个组氨酸残基以及天冬氨酸和谷氨酸残基组成。这些残基充当潜在的金属离子配体，并且 rCtFtn 同源模型预测该区域在蛋白质笼内突出。rCtFtn 还缺少位置 19 的保守酪氨酸残基。为了了解这些差异是否导致 rCtFtn 的亚铁氧化酶特性发生改变，我们通过定点突变在 rCtFtn 基因的位置 166 引入了一个终止密码子，并将位置 19 的丙氨酸残基替换为酪氨酸残基（rCtFtn166）。rCtFtn166 保留了被认为对该蛋白家族活性重要的经典序列。因此，我们期望 rCtFtn166 具有与该蛋白家族描述的相似性质。rCtFtn166 能够结合、氧化和储存铁；并且其活性被 Zn(II)抑制，如其他铁蛋白所描述的那样。然而，与野生型 rCtFtn 相比，rCtFtn166 的亚铁氧化酶活性和蛋白稳定性降低。对 Ala19Tyr rCtFtn 的分析表明，这种变化不会影响铁氧化的动力学。因此，这些结果表明 C 末端区域在亚铁氧化酶中心的活性和 rCtFtn 的稳定性中起重要作用。
