Levi S, Santambrogio P, Corsi B, Cozzi A, Arosio P
DIBIT Department of Biological and Technological Research, H. San Raffaele Scientific Institute, Milano, Italy.
Biochem J. 1996 Jul 15;317 ( Pt 2)(Pt 2):467-73. doi: 10.1042/bj3170467.
Iron is thought to enter the ferritin cavity via the three-fold channel, which is lined in its narrowest part by the residues Asp-131 and Glu-134. We describe here variants of human ferritins with active and inactive ferroxidase centres having Asp-131 and Glu-134 substituted with Ala and Ala or with Ile and Phe respectively. The two types of substitution had similar effects on ferritin functionality: (i) they decreased the amount of iron incorporated from Fe(II) solutions and decreased ferroxidase activity by about 50%; (ii) they inhibited iron incorporation from Fe(III) citrate in the presence of ascorbate; (iii) they resulted in loss of Fe and Tb binding sites; and (iv) they resulted in a marked decrease in the inhibition of iron oxidation by Tb (but not by Zn). In addition, it was found that substitution with Ala of Cys-130 and His-118, both of which face the three-fold channel, decreased the capacity of H-ferritin to bind terbium and to incorporate iron from Fe(III) citrate in the presence of ascorbate. The results indicate that: (i) in three-fold channels are the major sites of iron transfer into the cavity of H- and L-ferritins; (ii) at least two metal binding sites are located on the channels which play an active role in capturing and transferring iron into the cavity; and (iii) the permeability of the channel is apparently not affected by the hydrophilicity of its narrowest part. In addition, it is proposed that iron incorporation from Fe(III) citrate complexes in the presence of ascorbate is a reliable, and possibly more physiological, approach to the study of ferritin functionality.
铁被认为是通过三重通道进入铁蛋白腔的，该通道最窄处由天冬氨酸-131和谷氨酸-134残基排列。我们在此描述了人类铁蛋白的变体，其具有活性和无活性的铁氧化酶中心，其中天冬氨酸-131和谷氨酸-134分别被丙氨酸和丙氨酸或异亮氨酸和苯丙氨酸取代。这两种取代类型对铁蛋白功能有相似的影响：（i）它们减少了从Fe（II）溶液中掺入的铁量，并使铁氧化酶活性降低了约50%；（ii）在抗坏血酸存在下，它们抑制了柠檬酸铁（III）中铁的掺入；（iii）它们导致铁和铽结合位点的丧失；（iv）它们导致铽（而非锌）对铁氧化的抑制作用显著降低。此外，还发现面对三重通道的半胱氨酸-130和组氨酸-118被丙氨酸取代后，H-铁蛋白在抗坏血酸存在下结合铽和从柠檬酸铁（III）中掺入铁的能力降低。结果表明：（i）三重通道是铁进入H-和L-铁蛋白腔的主要部位；（ii）通道上至少有两个金属结合位点，它们在捕获和将铁转移到腔内发挥积极作用；（iii）通道的通透性显然不受其最窄部分亲水性的影响。此外，有人提出，在抗坏血酸存在下从柠檬酸铁（III）复合物中掺入铁是研究铁蛋白功能的一种可靠且可能更符合生理的方法。
