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Trapping and Electron Paramagnetic Resonance Characterization of the 5′dAdo• Radical in a Radical S-Adenosyl Methionine Enzyme Reaction with a Non-Native Substrate
ACS Central Science ( IF 18.2 ) Pub Date : 2019-09-25 , DOI: 10.1021/acscentsci.9b00706
Richard I. Sayler 1 , Troy A. Stich 1 , Sumedh Joshi 2 , Nicole Cooper 1 , Jared T. Shaw 1 , Tadhg P. Begley 2 , Dean J. Tantillo 1 , R. David Britt 1
Affiliation  

S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5′-deoxyadenosyl radical (5′dAdo), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5′dAdo radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe–S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)2CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe–Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the CαH-NH2 with a CH2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C2═C3 alkene substitution relative to HPPA’s single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5′dAdo radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C–H bond dissociation free energy as 5′dAdo, providing a possible explanation for our ability to trap an appreciable fraction of 5′dAdo in this specific rSAM reaction. The radical’s EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5′dAdo radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5′dAdo radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.

中文翻译:

非天然底物在自由基S-腺苷甲硫氨酸酶反应中5'dAdo 自由基的俘获和电子顺磁共振表征

S-腺苷甲硫氨酸(SAM)在自由基SAM(rSAM)酶的超家族中用作[4Fe-4S]结合的辅因子,其中[4Fe-4S] -SAM部分的单电子还原导致均质裂解的š -adenosyl甲硫氨酸产生5'-脱氧腺苷基(5'dAdo ),一种有效的H-原子抽象的。HydG是该rSAM家族的成员,使用5'dAdo 自由基裂解其底物酪氨酸,产生与第二HydG Fe-S簇的唯一Fe位点结合的CO和CN,最终产生单核有机金属Fe- l-半胱氨酸-(CO)2CN络合物作为[Fe-Fe]氢化酶催化H簇生物组装中的中间体。在这里,我们报告使用非天然酪氨酸底物类似物,以进一步探查HydG的初始自由基化学。一种这样的非天然底物是4-羟基苯基丙酸(HPPA),其缺乏酪氨酸的氨基,替换C α H-NH 2与CH 2在C2位。电子顺磁共振(EPR)研究表明,在HydG反应中,自然丰度和13 C2-HPPA生成了一个强而相对稳定的自由基,自旋密度位于C2处。这些结果使我们尝试使用氧化度更高的非天然底物香豆酸(其碳原子数为2)进行平行实验。相对于HPPA单键的C 3烯烃取代。有趣的是,HydG与顺式对香豆酸异构体的反应导致了新的自由基EPR信号的捕获,并且使用顺式对香豆酸和同位素标记的SAM进行的EPR研究表明,我们第一次被捕获并通过使用这种特定的非天然底物顺式-对-香豆酸,在实际的rSAM酶反应中表征了5'dAdo 自由基。密度泛函理论热力学计算表明,顺式-对香豆酸具有大约相同的C-H键离解自由能5'dAdo ,为我们在此特定rSAM反应中捕获相当一部分5'dAdo •的能力提供了可能的解释。该自由基的EPR线形及其随着SAM同位素取代而发生的变化与最近通过另一个rSAM酶丙酮酸甲酸酯裂解酶激活酶中预先还原的[4Fe-4S] -SAM中心进行低温光解而产生的5'dAdo 自由基的自由基几乎相同。,进一步支持了我们的任务,即我们已经通过明智地选择非天然底物来适当调节相对自由基自由能,从而确实在自由基SAM酶促反应中捕获并表征了5'dAdo 自由基。
更新日期:2019-11-28
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