Molecular Design of Metalloproteins

芳香族羟基化是进行的硬氧化之一，并且我们设计了一种肌红蛋白突变体，其能够基于以下策略氧化芳香环：1）在苯丙氨酸43的位置处引入底物色氨酸。2）将组氨酸64替换为酒石酸，以允许突变体与过氧化氢快速反应。同时，缬氨酸68也被异亮氨酸取代，以允许过氧化氢容易地进入活性位点。通过ESI-TOF质量测量监测蛋白质的氧化修饰。加入1 eq.过氧化氢的加入使分子量增加了16 Da。进一步加入过氧化氢得到二级产物，其质量增加32 Da。在蛋白质消化后，包括W 43的片段被纯化并通过MALDI TOF质谱和NMR表征。改性的残余物是具有6-羟基的W 43。这是通过化学计量量的过氧化氢进行芳族羟基化的第一个实例。此外，次级产物被表征为色氨酸43的2，6-二氧代衍生物。

The aromatic hydroxylation is one of the hard oxidations to proceed and we have designed a myoglobin mutant which is able to oxidize aromatic ring on the basis of the following strategies 1)Introduction of the substrate, tryptophan, at the position of phenyl alanine 43.2)Replacement of histidine 64 by asparitic acid to allow rapid reaction of the mutant with hydrogen peroxide. At the same time, Valine 68 was also replaced by isoleucine to allow hydrogen peroxide readily accessing to the active site, The oxidative modification of the protein was monitored by ESI-TOF mass measurements. Upon the addition of 1 eq. of hydrogen peroxide, 16Da increase of the molecular weight was observed. Further addition of hydrogen peroxide afforded secondary product exhibiting 32Da increase of its mass. After the digestion of the protein, a fragment including W43 was purified and characterized by MALDI TOF mass and NMR. The modified residue was W43 having 6-hydroxide. This is the first example of aromatic hydroxylation by a stoichiometric amount of hydrogen peroxide. In addition, the secondary product was characterized as 2,6-dioxo-derivative of tryptophan 43.