Outer-Sphere Effects on Reduction Potentials of Copper Sites in Proteins: The Curious Case of High Potential Type 2 C112D/M121E Pseudomonas aeruginosa Azurin

Redox and spectroscopic (electronic absorption, multifrequency electron paramagnetic resonance (EPR), and X-ray absorption) properties together with X-ray crystal structures are reported for the type 2 CuII C112D/M121E variant of Pseudomonas aeruginosa azurin. The results suggest that CuII is constrained from interaction with the proximal glutamate; this structural frustration implies a “rack” mechanism for the 290 mV (vs NHE) reduction potential measured at neutral pH. At high pH (∼9), hydrogen bonding in the outer coordination sphere is perturbed to allow axial glutamate ligation to CuII, with a decrease in potential to 119 mV. These results highlight the role played by outer-sphere interactions, and the structural constraints they impose, in determining the redox behavior of transition metal protein cofactors.

本研究报道了铜绿假单胞菌天青蛋白（Pseudomonas aeruginosa azurin）C112D/M121E突变体的2型铜(II)的氧化还原与光谱学性质，涵盖电子吸收光谱、多频电子顺磁共振（electron paramagnetic resonance, EPR）及X射线吸收光谱，同时同步提供其X射线晶体结构数据。实验结果表明，该铜(II)中心无法与近端谷氨酸残基发生相互作用；这种结构受挫现象暗示，在中性pH条件下测得的290 mV（相对于标准氢电极NHE）还原电位，其产生机制为“支架（rack）机制”。在高pH（约9）环境中，配位外层的氢键作用受到扰动，使得轴向谷氨酸残基可与铜(II)中心配位，此时还原电位降至119 mV。本研究结果凸显了配位外层相互作用及其所施加的结构约束，在决定过渡金属蛋白辅因子氧化还原行为中所发挥的关键作用。