Molecular Design of Metalloproteins

芳香羟基化是最难进行的氧化之一，我们设计了一个能够氧化芳香环的肌红蛋白突变体，基于以下策略：1)在苯基丙氨酸位置引入底物色氨酸；3.2)用天冬氨酸取代组氨酸64，使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，使过氧化氢容易进入活性位点，通过ESI-TOF质量测量来监测蛋白质的氧化修饰。加入1等量的过氧化氢后，其分子量增加16Da。进一步加入双氧水，二次产物质量增加32Da。蛋白质消化后，纯化了包含W43的片段，并通过MALDI TOF质量和NMR对其进行了表征。改性后的残渣为含有6-氢氧根的W43。这是第一个用化学计量量的过氧化氢进行芳香羟基化反应的例子。二级产物为色氨酸43的2,6-二氧基衍生物。

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.