Aubert C, Vos M H, Mathis P, Eker A P, Brettel K
Section de Bioénergétique (CNRS URA 2096), CEA Saclay, Gif-sur-Yvette, France.
Nature. 2000 Jun 1;405(6786):586-90. doi: 10.1038/35014644.
Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.
氨基酸自由基在许多酶促反应中起着关键作用。催化作用通常涉及蛋白质内部自由基特征的转移，如在I类核糖核苷酸还原酶中，自由基转移距离超过35埃，从一个酪氨酸自由基转移到一个半胱氨酸。目前存在争议的是，这种长程转移是通过电子转移，随后质子释放以产生中性自由基，还是通过氢原子转移，即电子和质子同时转移。后一种机制避免了在低介电常数蛋白质中形成电荷的能量成本，但与电子转移相比，它对结构变化的耐受性较差。现有的实验数据并不能明确区分这些提议。我们使用时间分辨吸收光谱研究了大肠杆菌DNA光解酶的光激活机制（黄素腺嘌呤二核苷酸辅因子的光诱导还原）。在这里我们表明，激发态黄素腺嘌呤二核苷酸自由基在30皮秒内从附近的色氨酸提取一个电子。在随后沿着三个色氨酸链进行电子转移后，最远端的色氨酸（作为阳离子自由基）在约300纳秒内向溶剂释放一个质子，表明电子转移发生在质子解离之前。类似的过程可能发生在类光解酶蓝光受体中。
