Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase
Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase
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DOI：
10.1074/jbc.m112.438796
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发表时间：
2013-04-05
影响因子：
4.8
通讯作者：
Haumann, Michael
中科院分区：
文献类型：
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作者：
Sigfridsson, Kajsa G. V.;Chernev, Petko;Haumann, Michael
原型双核金属辅因子与不同的代谢构成了一类O-2-活化催化剂在许多酶，如核糖核苷酸还原酶。需要可靠的结构来解开反应机制。然而，蛋白质晶体学数据可能会受到X射线光还原（XRP）的影响。我们使用X射线吸收光谱研究了沙眼衣原体核糖核苷酸还原酶R2亚基中Fe（III）Fe（III）和Mn（III）-Fe（III）位点的XPR。快速和双相X-射线光还原动力学在20和80 K的两种辅因子类型建议顺序形成（III，II）和（II，II）物种和类似的氧化还原电位的铁和锰网站。与晶体学中典型的X射线剂量相比，在这些研究中，（II，II）态在< 1 s内达到。第一球金属协调和金属-金属距离不同后，在室温下的化学还原和XPR后，在低温下，证实了从密度泛函理论计算的模型结构。然而，XPR诱导的（II，II）态中的金属间距离与R2晶体结构相似。因此，最初氧化的R2型蛋白质的晶体数据主要包含光还原（II，II）辅因子，偏离天然结构的功能在O-2激活，解释观察到的可变金属连接基序。这种情况可以通过新的飞秒自由电子激光蛋白质晶体学技术来补救。
Prototypic dinuclear metal cofactors with varying metallation constitute a class of O-2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III) Fe(III) and Mn(III)-Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III, II) and (II, II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II, II) states are reached in < 1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II, II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II, II) cofactors, which deviate from the native structures functional in O-2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.