Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase
Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase
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DOI：
10.1074/jbc.m604734200
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发表时间：
2007-02-16
影响因子：
4.8
通讯作者：
Weber, Stefan
中科院分区：
文献类型：
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作者：
Schleicher, Erik;Hitomi, Kenichi;Weber, Stefan
(6-4)光解酶催化含有（6-4）光产物的UV损伤DNA的光依赖性修复。酶的蓝光激发产生中性FAD自由基FADH（中心点），据信它是在酶促DNA修复过程中瞬时形成的。本文用光学光谱、电子顺磁共振和脉冲电子核双共振谱研究了（6-4）光裂合酶。FADH（中心点）的选定质子超精细偶联，即H-8 α和H-1 '的偶联的表征，产生了DNA底物预期结合位点处的微极性信息。在非洲爪蟾（6-4）光裂合酶中，超精细偶联的位移作为由点突变和pH变化诱导的结构修饰的函数，区分了两个高度保守的组氨酸（His（354）和His（358））的质子化状态。这些被提议催化氧杂环丁烷中间体的形成，该中间体先于光引发的DNA修复。结果表明，在pH 9.5时，酶修复活性最高，His 358被去质子化，而His（354）被质子化。因此，后者可能是引发氧杂环丁烷从（6-4）光产物形成的质子供体。
(6-4) photolyase catalyzes the light-dependent repair of UV-damaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH(center dot), which is believed to be transiently formed during the enzymatic DNA repair. Here (6-4) photolyase has been examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. Characterization of selected proton hyperfine couplings of FADH(center dot), namely those of H-8 alpha and H-1', yields information on the micropolarity at the site where the DNA substrate is expected to bind. Shifts in the hyperfine couplings as a function of structural modifications induced by point mutations and pH changes distinguish the protonation states of two highly conserved histidines, His(354) and His(358), in Xenopus laevis (6-4) photolyase. These are proposed to catalyze formation of the oxetane intermediate that precedes light-initiated DNA repair. The results show that at pH 9.5, where the enzymatic repair activity is highest, His 358 is deprotonated, whereas His(354) is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct.