Nilsen-Moe Astrid, Reinhardt Clorice R, Huang Ping, Agarwala Hemlata, Lopes Rosana, Lasagna Mauricio, Glover Starla, Hammes-Schiffer Sharon, Tommos Cecilia, Hammarström Leif
Department of Chemistry, Ångström Laboratory, Uppsala University Box 523 75120 Uppsala Sweden
Department of Molecular Biophysics and Biochemistry, Yale University New Haven CT 06520 USA.
Chem Sci. 2024 Jan 25;15(11):3957-3970. doi: 10.1039/d3sc05450k. eCollection 2024 Mar 13.
The proton-coupled electron transfer (PCET) reactions of tyrosine (Y) are instrumental to many redox reactions in nature. This study investigates how the local environment and the thermodynamic properties of Y influence its PCET characteristics. Herein, 2- and 4-mercaptophenol (MP) are placed in the well-folded αC protein (forming 2MP-αC and 4MP-αC) and oxidized by external light-generated [Ru(L)] complexes. The resulting neutral radicals are long-lived (>100 s) with distinct optical and EPR spectra. Calculated spin-density distributions are similar to canonical Y˙ and display very little spin on the S-S bridge that ligates the MPs to C inside the protein. With 2MP-αC and 4MP-αC we probe how proton transfer (PT) affects the PCET rate constants and mechanisms by varying the degree of solvent exposure or the potential to form an internal hydrogen bond. Solution NMR ensemble structures confirmed our intended design by displaying a major difference in the phenol OH solvent accessible surface area (≤∼2% for 2MP and 30-40% for 4MP). Additionally, 2MP-C is within hydrogen bonding distance to a nearby glutamate (average O-O distance is 3.2 ± 0.5 Å), which is suggested also by quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations. Neither increased exposure of the phenol OH to solvent (buffered water), nor the internal hydrogen bond, was found to significantly affect the PCET rates. However, the lower phenol p values associated with the MP-αC proteins compared to αY provided a sufficient change in PT driving force to alter the PCET mechanism. The PCET mechanism for 2MP-αC and 4MP-αC with moderately strong oxidants was predominantly step-wise PTET for pH values, but changed to concerted PCET at neutral pH values and below when a stronger oxidant was used, as found previously for αY. This shows how the balance of ET and PT driving forces is critical for controlling PCET mechanisms. The presented results improve our general understanding of amino-acid based PCET in enzymes.
酪氨酸（Y）的质子耦合电子转移（PCET）反应对自然界中的许多氧化还原反应至关重要。本研究调查了Y的局部环境和热力学性质如何影响其PCET特性。在此，将2-巯基苯酚和4-巯基苯酚（MP）置于折叠良好的αC蛋白中（形成2MP-αC和4MP-αC），并通过外部光生[Ru(L)]配合物进行氧化。产生的中性自由基寿命很长（>100 s），具有独特的光学和电子顺磁共振（EPR）光谱。计算得到的自旋密度分布与典型的Y˙相似，并且在将MP与蛋白质内部的C连接的S-S桥上显示出很少的自旋。利用2MP-αC和4MP-αC，我们通过改变溶剂暴露程度或形成内部氢键的可能性来探究质子转移（PT）如何影响PCET速率常数和机制。溶液核磁共振（NMR）集合结构通过显示酚羟基溶剂可及表面积的显著差异（2MP时≤约2%，4MP时为30 - 40%）证实了我们预期的设计。此外，2MP-C与附近的谷氨酸处于氢键距离内（平均O-O距离为3.2±0.5 Å），量子力学/分子力学（QM/MM）分子动力学模拟也表明了这一点。未发现酚羟基对溶剂（缓冲水）的暴露增加或内部氢键会显著影响PCET速率。然而，与αY相比，MP-αC蛋白的酚p值较低，这在PT驱动力方面提供了足够的变化，从而改变了PCET机制。对于2MP-αC和4MP-αC，在pH值下，与中等强度氧化剂反应时，PCET机制主要是分步PTET，但当使用更强的氧化剂时，在中性及以下pH值时会转变为协同PCET，这与之前αY的情况相同。这表明了电子转移（ET）和质子转移（PT）驱动力的平衡对于控制PCET机制至关重要。所呈现的结果增进了我们对酶中基于氨基酸的PCET的总体理解。
