Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in Fe IV =O Formation
Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in Fe IV =O Formation
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系列飞秒零剂量晶体学捕获染料脱色过氧化物酶中的无水远端血红素位点，揭示精氨酸在 Fe IV =O 形成中的催化作用
DOI：
10.1002/ange.202008622
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发表时间：
2020
影响因子：
--
通讯作者：
Lucic M
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文献类型：
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作者：
Lucic M
从零剂量X射线结晶学获得金属中心完整的氧化还原态结构可以促进我们对金属酶机理的理解。在染料脱色的血红素过氧化物酶(DYP)中，远端的血红素残基天冬氨酸和精氨酸在过氧化氢异解形成催化中间化合物 I(FeIV=O和一个卟啉阳离子自由基)中的作用存在争议。用系列飞秒X射线结晶学(SFX)测定了B型DyP的FeIII和FeIV=O氧化还原态的原始结构。这些结构揭示了远端无水的血红素位点，与天冬酰胺的存在一起，暗示了远端精氨酸作为催化碱的使用。诱变和动力学研究相结合证实了这一作用。因此，我们的SFX方法提供了独特的洞察力，了解如何调整DYP的远端血红素位置以选择天冬氨酸或精氨酸来提高过氧化氢异解的速度。
Obtaining structures of intact redox states of metal centers derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV=O and a porphyrin cation radical). Using serial femtosecond X‐ray crystallography (SFX), we have determined the pristine structures of the FeIIIand FeIV=O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.