Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, United States.
Department of Chemistry, Boston University, Boston, Massachusetts 02215, United States.
J Am Chem Soc. 2021 Sep 1;143(34):13835-13844. doi: 10.1021/jacs.1c06268. Epub 2021 Aug 23.
The diversity of the reactions catalyzed by radical -adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM enzymes, the radical quenching step is thought to involve an electron transfer to or from an auxiliary 4Fe-4S cluster in or adjacent to the active site. However, experimental demonstration of such functions remains limited. As a representative member of this family, MoaA has one radical SAM cluster ([4Fe-4S]) and one auxiliary cluster ([4Fe-4S]), and catalyzes a unique 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cHGTP) in the molybdenum cofactor (Moco) biosynthesis. Here, we report a mechanistic investigation of the radical quenching step in MoaA, a chemically challenging reduction of 3',8-cyclo-GTP-N7 aminyl radical. We first determined the reduction potentials of [4Fe-4S] and [4Fe-4S] as -510 mV and -455 mV, respectively, using a combination of protein film voltammogram (PFV) and electron paramagnetic resonance (EPR) spectroscopy. Subsequent Q-band EPR characterization of 5'-deoxyadenosine C4' radical (5'-dA-C4'•) trapped in the active site revealed isotropic exchange interaction (∼260 MHz) between 5'-dA-C4'• and [4Fe-4S], suggesting that [4Fe-4S] is in the reduced (1+) state during the catalysis. Together with density functional theory (DFT) calculation, we propose that the aminyl radical reduction proceeds through a proton-coupled electron transfer (PCET), where [4Fe-4S] serves as an electron donor and R17 residue acts as a proton donor. These results provide detailed mechanistic insights into the radical quenching step of radical SAM enzyme catalysis.
自由基-腺苷-L-甲硫氨酸（SAM）酶催化的反应多样性至少部分是通过各种机制来猝灭其自由基中间体实现的。在 SPASM-twitch 家族中，自由基 SAM 酶最大的家族，自由基猝灭步骤被认为涉及电子从辅助 4Fe-4S 簇转移到活性位点内或附近，或反之亦然。然而，对这些功能的实验证明仍然有限。作为这个家族的代表成员，MoaA 有一个自由基 SAM 簇（[4Fe-4S]）和一个辅助簇（[4Fe-4S]），并催化钼辅因子（Moco）生物合成中 GTP 的独特 3'，8-环化生成 3'，8-环-7,8-二氢-GTP（3'，8-cHGTP）。在这里，我们报道了 MoaA 中自由基猝灭步骤的机制研究，这是一个具有挑战性的 3'，8-环化-GTP-N7 氨自由基的化学还原。我们首先使用蛋白质膜伏安法（PFV）和电子顺磁共振（EPR）光谱相结合，确定了[4Fe-4S]和[4Fe-4S]的还原电位分别为-510 mV 和-455 mV。随后在活性位点中捕获 5'-脱氧腺苷 C4'自由基（5'-dA-C4'•）的 Q 波段 EPR 特征表明 5'-dA-C4'•与[4Fe-4S]之间存在各向同性交换相互作用（约 260 MHz），表明[4Fe-4S]在催化过程中处于还原（1+）态。结合密度泛函理论（DFT）计算，我们提出氨自由基还原通过质子耦合电子转移（PCET）进行，其中[4Fe-4S]作为电子供体，R17 残基作为质子供体。这些结果为自由基 SAM 酶催化的自由基猝灭步骤提供了详细的机制见解。
