The ferroxidase reaction of ferritin reveals a diferric μ-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins
The ferroxidase reaction of ferritin reveals a diferric μ-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins
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DOI：
10.1021/bi990095l
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发表时间：
1999-04-27
期刊：
影响因子：
2.9
通讯作者：
Loehr, TM
中科院分区：
文献类型：
--
作者：
Möenne-Loccoz, P;Krebs, C;Loehr, TM
铁蛋白是普遍存在的蛋白质，可通过 Fe2+ 的氧化（铁氧化）浓缩、储存细胞内的铁并对其进行解毒，然后进行易位和水解，形成大型无机矿物核心。对铁蛋白的一系列诱变、动力学和光谱研究得出了氧化/易位路径涉及二铁蛋白位点的建议。最近的停流吸收和快速冷冻淬灭穆斯堡尔研究已确定单一过氧二铁物种是重组青蛙 M 铁蛋白在快速亚铁氧化过程中形成的初始瞬时中间体[Pereira, S, A., Small, W,, Krebs, C,, Tavares, P,, Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]，为了进一步表征这种瞬态中间体并明确建立过氧二铁分配，使用快速冷冻猝灭来捕获初始中间体以进行共振拉曼研究。观察到该中间体的离散振动模式，表明单个发色团处于均匀状态，与穆斯堡尔的结论一致。 851 cm(-1)处的频率被指定为结合过氧化物的nu(O-O)，485和499 cm(-1)处的频率对分别归因于Fe-O-2-Fe的nu(s)和nu(as)。这些拉曼带的同位素敏感性可将发色团鉴定为 mu-1,2 桥接的过氧化二铁。在大肠杆菌核糖核苷酸还原酶 R2 亚基突变体和化学还原的 Delta(9) 硬脂酰酰基载体蛋白去饱和酶 (Delta 9D) 中也检测到了类似的过氧二铁中间体，但相反，铁蛋白中间体被从天然蛋白的真实反应途径中捕获。这些过氧化物种类的拉曼特征的差异归因于 Fe-O-O-Fe 角的变化，并且可能与铁是保留在催化中心还是作为氧化产物释放有关。
Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mossbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S, A., Small, W,, Krebs, C,, Tavares, P,, Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876], To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mossbauer conclusions. The frequency at 851 cm(-1) is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm(-1) is attributed, respectively, to nu(s) and nu(as) of Fe-O-2-Fe. Identification of the chromophore as a mu-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta(9) stearoyl-acyl carrier protein desaturase (Delta 9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.