Levi S, Corsi B, Rovida E, Cozzi A, Santambrogio P, Albertini A, Arosio P
Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
J Biol Chem. 1994 Dec 2;269(48):30334-9.
Ferritins are 24-mer proteins which store and detoxify intracellular iron. Mammalian ferritins are made of two subunit types, the H- and L-chains, with different functional specificity. The H-chain has a metal-binding site (the ferroxidase center) which confers ferroxidase activity to the protein and accelerates iron incorporation. In the L-chain the center is substituted by a salt bridge. We performed several site-directed mutageneses in the L-chain with the aim to construct the center and confer ferroxidase activity to the protein. Most variants were insoluble and did not refold into homopolymers, probably due to electrostatic repulsion introduced by the substitutions. However, they formed hybrids when they were renatured together with the L- or H-chains. The heteropolymers made of 90% L-chain and 10% of an L-variant with all the ligand residues of the H-chain center had 25-30% of the ferroxidase activity of the H-chain homopolymer. This corresponds to the activity of an H/L heteropolymer with 7% H-chain. It is concluded that: (i) it is possible to construct a ferroxidase center in the L-chain with an activity equivalent to that of the H-chain, (ii) the residues of the center interfere with the folding/assembly of the L-, but not of the H-chain, (iii) heteropolymers can be made even between ferritin subunits with large differences of refolding rates.
铁蛋白是一种由24个亚基组成的蛋白质，负责储存细胞内的铁并使其解毒。哺乳动物铁蛋白由两种亚基类型组成，即H链和L链，具有不同的功能特异性。H链具有一个金属结合位点（铁氧化酶中心），该位点赋予蛋白质铁氧化酶活性并加速铁的掺入。在L链中，该中心被一个盐桥取代。我们在L链中进行了多次定点诱变，目的是构建该中心并赋予蛋白质铁氧化酶活性。大多数变体不溶，无法重新折叠成同聚物，这可能是由于取代引入的静电排斥所致。然而，当它们与L链或H链一起复性时，它们形成了杂合物。由90%的L链和10%的具有H链中心所有配体残基的L变体组成的杂聚物具有H链同聚物25%-30%的铁氧化酶活性。这相当于具有7%H链的H/L杂聚物的活性。结论如下：（i）有可能在L链中构建一个活性与H链相当的铁氧化酶中心；（ii）该中心的残基会干扰L链的折叠/组装，但不会干扰H链的；（iii）即使在重折叠速率差异很大的铁蛋白亚基之间也可以形成杂聚物。
