Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping
Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping
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DOI：
10.1021/jacs.9b05715
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发表时间：
2019-11-06
影响因子：
15
通讯作者：
Crane，Brian R.
中科院分区：
文献类型：
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作者：
Yee，Estella F.;Dzikovski，Boris;Crane，Brian R.
瞬时酪氨酸和色氨酸自由基在光系统（PS）II、核糖核苷酸还原酶（RNR）、光裂合酶和许多其他蛋白质的电子转移（ET）反应中起关键作用。然而，Tyr和Trp在功能上是不可互换的，并且控制它们的反应性的因素通常是不清楚的。细胞色素过氧化物酶（Cytochromecperoxidase，CcP）利用Trp 191·+自由基氧化还原的细胞色素c（Cytochromecperoxidase，Cc）。虽然Tyr 191取代也形成稳定的自由基，但它不支持从Cc快速ET。在这里，我们通过非天然氨基酸取代、改变ET驱动力和操纵Y191的质子环境来探索CcP Y191的氧化还原性质。更高的潜在氟酪氨酸残基略微增加ET速率，但仅将氢键供体添加到Tyr 191·（通过Leu 232 His或Glu）显著改变活性，使ET速率增加近30倍。ESR和ESEEM光谱、晶体学和pH依赖性ET动力学为His 232/Glu 232与Y191·形成氢键提供了强有力的证据。速率测量和快速冷冻淬火ESR光谱进一步揭示了自由基传播和C氧化的差异，支持在E232存在下增加的Y191·形式电位为200 mV。因此，Y191失活是由于Y191·+去质子化引起的电位下降。引入一个位置良好的碱基来接受和回馈一个氢键，使Tyr·电位升高到一个可以有效氧化Cc的范围。这些发现对PS II的YZ/YDradicals，RNR和隐花色素中的空穴跳跃以及用于远程ET反应的工程蛋白质具有影响。
Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and the factors controlling their reactivity are often unclear. Cytochromecperoxidase (CcP) employs a Trp191•+radical to oxidize reduced cytochrome c (Cc). Although a Tyr191 replacement also forms a stable radical, it does not support rapid ET fromCc. Here we probe the redox properties of CcP Y191 by non-natural amino acid substitution, altering the ET driving force and manipulating the protic environment of Y191. Higher potential fluorotyrosine residues increase ET rates marginally, but only addition of a hydrogen bond donor to Tyr191•(via Leu232His or Glu) substantially alters activity by increasing the ET rate by nearly 30-fold. ESR and ESEEM spectroscopies, crystallography, and pH-dependent ET kinetics provide strong evidence for hydrogen bond formation to Y191•by His232/Glu232. Rate measurements and rapid freeze quench ESR spectroscopy further reveal differences in radical propagation andCcoxidation that support an increased Y191•formal potential of ∼200 mV in the presence of E232. Hence, Y191 inactivity results from a potential drop owing to Y191•+deprotonation. Incorporation of a well-positioned base to accept and donate back a hydrogen bond upshifts the Tyr•potential into a range where it can effectively oxidizeCc. These findings have implications for the YZ/YDradicals of PS II, hole-hopping in RNR and cryptochrome, and engineering proteins for long-range ET reactions.