Davis Ian, Koto Teruaki, Liu Aimin
Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
React Oxyg Species (Apex). 2018 Jan;5(13):46-55. Epub 2018 Jan 1.
The di-heme enzyme, MauG, utilizes a high-valent, charge-resonance stabilized -Fe(IV) state to perform protein radical-based catalytic chemistry. Though the -Fe(IV) species is able to oxidize remote tryptophan residues on its substrate protein, it does not rapidly oxidize its own residues in the absence of substrate. The slow return of -Fe(IV) MauG to its resting di-ferric state occurs up to two intermediates, one of which has been previously proposed by Ma (Biochem J 2016; 473:1769) to be a methionine-based radical in a recent study. In this work, we pursue intermediates involved in the return of high-valent MauG to its resting state in the absence of the substrate by EPR spectroscopy and radical trapping. The -Fe(IV) MauG is shown by EPR, HPLC, UV-Vis, and high-resolution mass spectrometry to oxidize the trapping agent, 5,5-dimethyl-1-pyrroline -oxide (DMPO) to a radical species directly. Nitrosobenzene was also employed as a trapping agent and was shown to form an adduct with high-valent MauG species. The effects of DMPO and nitrosobenzene on the kinetics of the return to di-ferric MauG were both investigated. This work eliminates the possibility that a MauG-based methionine radical species accumulates during the self-reduction of -Fe(IV) MauG.
双血红素酶MauG利用一种高价、电荷共振稳定的 -Fe(IV) 状态来进行基于蛋白质自由基的催化化学反应。尽管 -Fe(IV) 物种能够氧化其底物蛋白上远端的色氨酸残基，但在没有底物的情况下它不会快速氧化自身的残基。-Fe(IV) MauG缓慢恢复到其静止的二价铁状态会经过多达两个中间体，其中一个中间体在最近的一项研究中被Ma（《生物化学杂志》2016年；473:1769）提出是基于甲硫氨酸的自由基。在这项工作中，我们通过电子顺磁共振光谱（EPR）和自由基捕获来研究在没有底物的情况下参与高价MauG恢复到其静止状态的中间体。EPR、高效液相色谱（HPLC）、紫外可见光谱（UV-Vis）和高分辨率质谱表明，-Fe(IV) MauG直接将捕获剂5,5-二甲基-1-吡咯啉 -氧化物（DMPO）氧化为自由基物种。亚硝基苯也被用作捕获剂，并被证明与高价MauG物种形成加合物。我们还研究了DMPO和亚硝基苯对MauG恢复到二价铁状态动力学的影响。这项工作排除了在 -Fe(IV) MauG自我还原过程中基于MauG的甲硫氨酸自由基物种积累的可能性。
