Spectroscopic and DFT Studies of Second-Sphere Variants of the Type 1 Copper Site in Azurin: Covalent and Nonlocal Electrostatic Contributions to Reduction Potentials

详细信息    查看全文

• 作者：Ryan G. Hadt ; Ning Sun ; Nicholas M. Marshall ; Keith O. Hodgson ; Britt Hedman ; Yi Lu ; Edward I. Solomon
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：October 10, 2012
• 年：2012
• 卷：134
• 期：40
• 页码：16701-16716
• 全文大小：666K
• 年卷期：v.134,no.40(October 10, 2012)
• ISSN：1520-5126

文摘

The reduction potentials (E⁰) of type 1 (T1) or blue copper (BC) sites in proteins and enzymes with identical first coordination spheres around the redox active copper ion can vary by 400 mV. Here, we use a combination of low-temperature electronic absorption and magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, and S聽K-edge X-ray absorption spectroscopies to investigate a series of second-sphere variants鈥擣114P, N47S, and F114N in Pseudomonas aeruginosa azurin鈥攚hich modulate hydrogen bonding to and protein-derived dipoles nearby the Cu鈥揝(Cys) bond. Density functional theory calculations correlated to the experimental data allow for the fractionation of the contributions to tuning E⁰ into covalent and nonlocal electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly nonlocal electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from nonlocal electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long-range protein/active interactions, while affording further insight into the second-sphere mechanisms available to the protein to tune the E⁰ of electron-transfer sites in biology.