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Abstract：
Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.
摘要：
染料脱色过氧化物酶（DyP）是血红素蛋白，与经典过氧化物酶相比具有不同的结构特性和底物特异性。这里,我们证明了来自极端耐辐射细菌耐放射球菌的DyP是，像其他一些同源物一样，在生理pH值下无活性。共振拉曼（RR）光谱证实，血红素在pH7.5时处于六配位低自旋（6cLS）状态，因此无法结合过氧化氢。在pH4.0时，酶的RR光谱揭示了高自旋和低自旋血红素态的共存，这证实了在较低pH下检测到的对H2O2的催化活性。与其他DyP的序列比对显示DrDyP在高度保守的GXXDG基序的第5位具有甲硫氨酸残基。为了分析甲硫氨酸的存在是否导致在高pH下缺乏活性，该残基被甘氨酸取代。UV-vis和RR光谱揭示所得DrDyPM190G在pH7.5时也处于6cLS自旋状态,因此甲硫氨酸不影响蛋白质的活性。DrDyP和DrDyPM190G的晶体结构,分辨率为2.20和1.53，分别,然而，揭示了有趣的见解。DrDyPM190G的高分辨率结构，在pH8.5下获得的，表明一个羟基和一个水分子在与血红素和催化天冬酰胺和精氨酸的氢键距离内。这种强配体最有可能阻止H2O2底物的结合，强化关于这个和其他DYP的生理底物的问题，以及可能引发羟基去除的可能事件，赋予DrDyP催化活性。
