Pereira A S, Small W, Krebs C, Tavares P, Edmondson D E, Theil E C, Huynh B H
Department of Physics, Emory University, Atlanta, Georgia 30322, USA.
Biochemistry. 1998 Jul 14;37(28):9871-6. doi: 10.1021/bi980847w.
Rapid freeze-quench (RFQ) Mössbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mössbauer parameters (DeltaEQ = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mössbauer method match those of a transient blue species (lambdamax = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.
采用快速冷冻淬灭（RFQ）穆斯堡尔光谱和停流吸收光谱法，利用重组（脱辅基）蛙M型铁蛋白监测铁蛋白铁氧化酶反应；通过RFQ方法，使用低铁负载量（36个Fe2+/铁蛋白分子）可以捕获初始瞬态铁物种。用穆斯堡尔方法直接观察并测量了铁氧化的双相动力学；大部分（85%）亚铁离子以约80 s-1的快速率被氧化，其余部分以约1.7 s-1的慢得多的速率被氧化。与Fe2+离子的快速氧化阶段同时，形成了一种单一的瞬态铁物种，其具有磁性特性（抗磁性基态）和穆斯堡尔参数（ΔEQ = 1.08±0.03 mm/s和δ = 0.62±0.02 mm/s），表明是一种反铁磁耦合的过氧二铁络合物。通过RFQ穆斯堡尔方法测量的这种瞬态双铁物种的形成和衰减速率，与通过停流吸光度测量确定的瞬态蓝色物种（λmax = 650 nm）的速率相匹配。因此，将瞬态有色物种归为同一过氧二铁中间体。其他研究人员在其他几种快速铁蛋白（H和M亚基类型）中也检测到了类似的瞬态有色物种，如人H型铁蛋白和大肠杆菌铁蛋白，这表明所有快速铁蛋白中铁蛋白铁氧化酶步骤的机制相似。过氧二铁络合物也是O2与可溶性甲烷单加氧酶（MMOH）羟化酶组分中的催化双铁中心以及大肠杆菌核糖核苷酸还原酶R2亚基的D84E突变体反应的早期中间体。蛙M型铁蛋白中形成的初始过氧二铁物种的动力学、光谱性质和纯度，证实了关于一个与MMOH和R2中的双铁中心结构同源的单一蛋白质位点参与铁蛋白铁氧化步骤的提议。
