Department of Chemistry, State University of New York, Potsdam, NY 13676, USA.
Metallomics. 2019 Apr 17;11(4):774-783. doi: 10.1039/c9mt00001a.
The excessively high and inconsistent literature values for Km,Fe and Km,O2 prompted us to examine the iron oxidation kinetics in ferritin, the major iron storage protein in mammals, and to determine whether a traditional Michaelis-Menten enzymatic behavior is obeyed. The kinetics of Fe(ii) oxidation and mineralization catalyzed by three different types of ferritins (recombinant human homopolymer 24H, HuHF, human heteropolymer ∼21H:3L, HL, and horse spleen heteropolymer ∼3.3H:20.7L, HosF) were therefore studied under physiologically relevant O2 concentrations, but also in the presence of excess Fe(ii) and O2 concentrations. The observed iron oxidation kinetics exhibited two distinct phases (phase I and phase II), neither of which obeyed Michaelis-Menten kinetics. While phase I was very rapid and corresponded to the oxidation of approximately 2 Fe(ii) ions per H-subunit, phase II was much slower and varied linearly with the concentration of iron(ii) cations in solution, independent of the size of the iron core. Under low oxygen concentration close to physiological, the iron uptake kinetics revealed a Michaelis-Menten behavior with Km,O2 values in the low μM range (i.e. ∼1-2 μM range). Our experimental Km,O2 values are significantly lower than typical cellular oxygen concentration, indicating that iron oxidation and mineralization in ferritin should not be affected by the oxygenation level of cells, and should proceed even under hypoxic events. A kinetic model is proposed in which the inhibition of the protein's activity is caused by bound iron(iii) cations at the ferroxidase center, with the rate limiting step corresponding to an exchange or a displacement reaction between incoming Fe(ii) cations and bound Fe(iii) cations.
文献中 Km、Fe 和 Km,O2 的数值过高且不一致，这促使我们研究铁蛋白（哺乳动物中主要的铁储存蛋白）中的铁氧化动力学，并确定其是否遵循传统的米氏酶动力学行为。因此，我们在生理相关的 O2 浓度下，以及在存在过量 Fe(ii)和 O2 浓度的情况下，研究了三种不同类型的铁蛋白（重组人同源单体 24H、HuHF、人异源单体 ∼21H:3L、HL 和马脾异源单体 ∼3.3H:20.7L、HosF）催化的 Fe(ii)氧化和矿化动力学。观察到的铁氧化动力学表现出两个明显的阶段（阶段 I 和阶段 II），都不符合米氏酶动力学。虽然阶段 I 非常快，对应于每个 H 亚基氧化约 2 个 Fe(ii)离子，但阶段 II 要慢得多，并且与溶液中 Fe(ii)阳离子的浓度呈线性关系，与铁核的大小无关。在接近生理的低氧浓度下，铁摄取动力学呈现出米氏酶行为，Km,O2 值在低 μM 范围内（即 1-2 μM 范围内）。我们的实验 Km,O2 值明显低于典型的细胞氧浓度，表明铁蛋白中铁的氧化和矿化不应受细胞氧合水平的影响，即使在缺氧事件下也应进行。提出了一种动力学模型，其中蛋白活性的抑制是由铁氧还蛋白中心结合的铁(iii)阳离子引起的，限速步骤对应于进入的 Fe(ii)阳离子和结合的 Fe(iii)阳离子之间的交换或取代反应。
