Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
Department of Chemistry, University of York, Heslington, York YO10 5DD, U.K.
J Am Chem Soc. 2023 Sep 20;145(37):20672-20682. doi: 10.1021/jacs.3c06607. Epub 2023 Sep 9.
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a Cu-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled ( = 1) Cu-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
加氧酶和过氧酶在其活性部位生成中间体，从而实现对底物中惰性 C-H 键的受控功能化，例如在甲烷到甲醇的酶促转化中。然而，为了成为可行的催化剂，这些酶还必须防止在偶联和非偶联周转期间发生的关键活性部位残基的氧化损伤。在此，我们使用停流光谱学、靶向突变、TD-DFT 计算、高能分辨率荧光检测 X 射线吸收光谱学和电子顺磁共振波谱学相结合的方法来研究两种瞬态中间体，它们共同构成了铜依赖性溶菌多糖单加氧酶（LPMO）活性部位中的内置保护途径。首先，在不存在底物的情况下，LPMO 用过氧化氢或过氧酸处理后，在铜组氨酸臂活性部位生成瞬态高价物种。我们提出这个中间体是一个 Cu-（组氨酸自由基），然后通过系间交叉反应与附近的酪氨酸残基反应，生成铁磁耦合的（ = 1）Cu-酪氨酸自由基对，从而使组氨酸臂活性部位恢复到其静止状态，并允许它通过还原重新进入催化循环。这个过程使酶具有在活性部位组氨酸残基“飞行中”最小化损伤的能力，从而在酶失活之前增加总的周转数，突出了氧化酶如何进化以保护自己免受非偶联周转期间有害的副反应的影响。
