Department of Chemistry, Stanford University, Stanford, CA 94305.
Department of Chemistry, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
Proc Natl Acad Sci U S A. 2021 Apr 13;118(15). doi: 10.1073/pnas.2022379118.
The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the Fe active site to form a highly reactive Fe = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-Fe = O intermediate and characterized its structure as a Fe-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary Fe active site before the O reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of Fe = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the Fe site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the Fe site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.
依赖蝶呤的非血红素铁酶羟化芳香族氨基酸，以执行神经递质的生物合成，从而维持大脑的正常功能。这些酶利用蝶呤辅因子和与 Fe 活性位点结合的芳香族氨基酸底物来激活氧，形成一种高反应性的 Fe = O 物种，从而引发底物氧化。在这项研究中，我们使用色氨酸羟化酶在动力学上生成了一个预-Fe = O 中间体，并通过吸收光谱、穆斯堡尔光谱、共振拉曼光谱和核共振振动光谱学将其结构表征为 Fe-过氧蝶呤物种。通过对氧反应前的蝶呤辅因子和色氨酸底物结合的三元 Fe 活性位点的平行表征（包括磁圆二色性光谱学），这些研究从实验上定义了 Fe = O 形成的机制，并证明了蝶呤上的羰基官能团在三元复合物和过氧中间体中都直接与 Fe 位点配位。反应坐标计算预测，由于蝶呤羰基与 Fe 位点的直接结合，氧活化势垒会降低 14 kcal/mol，因为这种相互作用为蝶呤辅因子向铁中心的有效电子转移提供了轨道途径。蝶呤辅因子的这种直接配位使依赖蝶呤的羟化酶具有生物学功能，并证明了辅因子依赖性非血红素铁酶氧活化的统一机制。
