MECHANISM OF ASSEMBLY OF THE TYROSYL RADICAL DINUCLEAR IRON CLUSTER COFACTOR OF RIBONUCLEOTIDE REDUCTASE
MECHANISM OF ASSEMBLY OF THE TYROSYL RADICAL DINUCLEAR IRON CLUSTER COFACTOR OF RIBONUCLEOTIDE REDUCTASE
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DOI：
10.1126/science.1650033
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发表时间：
1991-07-19
期刊：
影响因子：
56.9
通讯作者：
STUBBE, J
中科院分区：
文献类型：
--
作者：
BOLLINGER, JM;EDMONDSON, DE;STUBBE, J
大肠杆菌核糖核苷酸还原酶apoB2亚基与Fe2+和O2孵育产生天然B2，其中含有核苷酸还原所需的酪氨酸自由基-双核铁簇辅因子。采用停流吸收光谱和快速冷冻顺磁共振（EPR）光谱研究了该重构反应的化学机理。在反应中发现了两种新的中间产物。第一种材料具有以565纳米为中心的宽吸收带。根据已知的模型化学，该中间体被认为是一种多过氧异铁配合物。第二中间体表现出以360纳米为中心的宽吸收带和一个尖锐的各向同性EPR信号，其g = 2.00。当与Fe-57（2+）反应时，EPR信号展宽，表明中间体是铁偶联自由基。通过对反应中Fe2+与B2的比值变化以及中间产物的生成速率和衰变速率与酪氨酸自由基（. y122）的生成速率的比较表明，这两种中间产物都能生成。y122。这一结论得到了以下事实的支持：在缺乏可氧化Y122的突变体B2亚基（B2- y122f）中，这两种中间体的寿命都增加了。根据这些动力学和光谱数据，提出了反应的机理。与血红素-铁过氧化物酶、加氧酶和模式配合物催化的反应不同，重组反应似乎不涉及高价铁中间体。
Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with Fe-57(2+), this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.