Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer
Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer
复制标题
DOI：
10.1073/pnas.1521664113
复制
发表时间：
2016-02-02
影响因子：
11.1
通讯作者：
Poulos, Thomas L.
中科院分区：
文献类型：
--
作者：
Chreifi, Georges;Baxter, Elizabeth L.;Poulos, Thomas L.
过氧化物与过氧化物酶的反应将血红素铁从Fe（III）氧化为Fe（IV）=O，并将卟啉或芳族侧链氧化为阳离子自由基。X射线产生的水合电子迅速还原Fe（IV），因此需要使用许多晶体进行非常短的暴露，即使这样，也无法避免一些还原。新一代X射线自由电子激光器能够在十分之一的飞秒时间尺度上产生强烈的X射线，能够在不减少或X射线损伤的情况下进行结构测定。在这里，我们报告的1.5埃晶体结构的细胞色素c过氧化物酶（CCP）化合物I（CmpI）使用的数据与斯坦福大学线性相干光源（LCLS）。这种结构与以前的结构是一致的。特别重要的是活性位点水结构，其可以介导CmpI形成和Fe（IV）=O还原为Fe（III）-OH所需的质子转移反应。这些结构表明，水分子理想地定位于在铁连接的氧和活性位点催化His之间穿梭质子。因此，我们进行了计算和动力学研究，以探测Fe（IV）=O的还原。动力学溶剂同位素实验表明，在过氧化物酶限速步骤中，单个质子的转移是关键的，这很可能是Fe（IV）=O到Fe（III）OH的质子偶联还原。我们还发现催化His的pK（a）在CmpI中显著增加，表明该活性位点His是Fe（IV）=O还原为Fe（IV）-OH所需的质子来源。
The reaction of peroxides with peroxidases oxidizes the heme iron from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical. X-ray-generated hydrated electrons rapidly reduce Fe(IV), thereby requiring very short exposures using many crystals, and, even then, some reduction cannot be avoided. The new generation of X-ray free electron lasers capable of generating intense X-rays on the tenths of femtosecond time scale enables structure determination with no reduction or X-ray damage. Here, we report the 1.5-angstrom crystal structure of cytochrome c peroxidase (CCP) compound I (CmpI) using data obtained with the Stanford Linear Coherent Light Source (LCLS). This structure is consistent with previous structures. Of particular importance is the active site water structure that can mediate the proton transfer reactions required for both CmpI formation and reduction of Fe(IV)=O to Fe(III)-OH. The structures indicate that a water molecule is ideally positioned to shuttle protons between an iron-linked oxygen and the active site catalytic His. We therefore have carried out both computational and kinetic studies to probe the reduction of Fe (IV)=O. Kinetic solvent isotope experiments show that the transfer of a single proton is critical in the peroxidase rate-limiting step, which is very likely the proton-coupled reduction of Fe(IV)=O to Fe(III)OH. We also find that the pK(a) of the catalytic His substantially increases in CmpI, indicating that this active site His is the source of the proton required in the reduction of Fe(IV)=O to Fe(IV)-OH.