Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin
Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin
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DOI：
10.1021/acs.inorgchem.3c01365
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发表时间：
2023-07-10
影响因子：
4.6
通讯作者：
Lu，Yi
中科院分区：
文献类型：
--
作者：
Liu，Yiwei;Marshall，Nicholas M.;Lu，Yi
为了研究次级配位球对铜氧还蛋白中1型蓝铜(T1Cu)氧化还原电位(E°′)微调的影响，我们在铜绿假单胞菌天青蛋白(Az)的T1Cu中心的次级配位球中单独或组合引入了M13F、M44F和G116F突变。发现这些变体对 T1Cu 的 E°' 有不同的影响，其中 M13F Az 降低了 E°'，M44F Az 增加了 E°'，而 G116F Az 的影响可以忽略不计。此外，M13F和M44F突变的组合相对于WT-Az使E°'增加了26 mV，这非常接近每个突变对E°'的组合效应。此外，将 G116F 与 M13F 或 M44F 突变结合分别会产生负面和正面的协同效应。 M13F/M44F-Az、M13F/G116F-Az 和 M44F/G116F-Az 的晶体结构与 G116F-Az 的晶体结构相结合揭示了这些变化是由空间效应和铜结合 His117 残基周围氢键网络的微调引起的。从这项研究中获得的见解将为开发具有可调氧化还原特性的氧化还原活性蛋白迈出又一步，用于许多生物和生物技术应用。
In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°′) of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) fromPseudomonas aeruginosa. These variants were found to differentially influence theE°′ of T1Cu, with M13F Az decreasingE°′, M44F Az increasingE°′, and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increasesE°′ by 26 mV relative to WT-Az, which is very close to the combined effect ofE°′ by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.