Jameson Guy N L, Jin Weili, Krebs Carsten, Perreira Alice S, Tavares Pedro, Liu Xiaofeng, Theil Elizabeth C, Huynh Boi Hanh
Department of Physics, Emory University, Atlanta, Georgia 30322, USA.
Biochemistry. 2002 Nov 12;41(45):13435-43. doi: 10.1021/bi026478s.
The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F(peroxo), formed within 25 ms, which then leads to the release of H(2)O(2) and formation of ferric mineral precursors. The stoichiometric relationship between F(peroxo), H(2)O(2), and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H(2)O(2). By rapidly mixing apo M ferritin from frog, Fe(2+), and O(2) and allowing the reaction to proceed for 70 ms when F(peroxo) has reached its maximum accumulation, followed by spraying the reaction mixture into the H(2)O(2) assay solution, we were able to quantitatively determine the amount of H(2)O(2) produced during the decay of F(peroxo). The correlation between the amount of H(2)O(2) released with the amount of F(peroxo) accumulated at 70 ms determined by Mössbauer spectroscopy showed that F(peroxo) decays into H(2)O(2) with a stoichiometry of 1 F(peroxo):H(2)O(2). When the decay of F(peroxo) was monitored by rapid freeze-quench Mössbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F(peroxo). This observed parallel formation of multiple products (H(2)O(2), diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F(peroxo)) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.
在H型铁蛋白中心腔内引发氢氧化铁矿物核心形成的催化步骤涉及在每个24个亚基中发现的双核位点（铁氧化酶位点）上，亚铁离子被分子氧快速氧化（铁氧化酶反应）。先前的研究人员表明，铁氧化酶反应的第一个可检测反应中间体是在25毫秒内形成的双铁过氧中间体F(peroxo)，然后它会导致H₂O₂的释放和铁矿物前体的形成。F(peroxo)、H₂O₂和铁矿物前体之间的化学计量关系对于确定反应途径和机制至关重要，现在已经确定。为此，使用辣根过氧化物酶催化的分光光度法来测定H₂O₂。通过快速混合来自青蛙的脱辅基M铁蛋白、Fe²⁺和O₂，并在F(peroxo)达到其最大积累量时让反应进行70毫秒，然后将反应混合物喷入H₂O₂测定溶液中，我们能够定量测定F(peroxo)衰变过程中产生的H₂O₂的量。通过穆斯堡尔光谱法测定的在70毫秒时积累的F(peroxo)量与释放的H₂O₂量之间的相关性表明，F(peroxo)以1 F(peroxo):H₂O₂的化学计量比衰变为H₂O₂。当通过快速冷冻淬灭穆斯堡尔光谱法监测F(peroxo)的衰变时，发现多个双铁μ-氧/μ-羟基配合物和小的多核铁簇以与F(peroxo)衰变速率相同的速率常数形成。从单个前体（F(peroxo)）的衰变中观察到的多种产物（H₂O₂、双铁配合物和小的多核簇）的平行形成，为铁蛋白矿化提供了有用的机制见解，并证明了一个灵活的铁氧化酶位点。
