Characterization of the Copper(II) Binding Sites in Human Carbonic Anhydrase II

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• 作者：Whitnee L. Nettles ; He Song ; Erik R. Farquhar ; Nicholas C. Fitzkee ; Joseph P. Emerson
• 刊名：Inorganic Chemistry
• 出版年：2015
• 出版时间：June 15, 2015
• 年：2015
• 卷：54
• 期：12
• 页码：5671-5680
• 全文大小：452K
• ISSN：1520-510X

文摘

Human carbonic anhydrase (CA) is a well-studied, robust, mononuclear Zn-containing metalloprotein that serves as an excellent biological ligand system to study the thermodynamics associated with metal ion coordination chemistry in aqueous solution. The apo form of human carbonic anhydrase II (CA) binds 2 equiv of copper(II) with high affinity. The Cu²⁺ ions bind independently forming two noncoupled type II copper centers in CA (Cu_A and Cu_B). However, the location and coordination mode of the Cu_A site in solution is unclear, compared to the Cu_B site that has been well-characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we identified an N-terminal Cu²⁺ binding location and collected information on the coordination mode of the Cu_A site in CA, which is consistent with a four- to five-coordinate N-terminal Cu²⁺ binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-amino terminal Cu²⁺ and Ni²⁺ and copper(II)-尾-amyloid complexes. Additionally, we report a more detailed analysis of the thermodynamics associated with copper(II) binding to CA. Although we are still unable to fully deconvolute Cu²⁺ binding data to the high-affinity Cu_A site, we derived pH- and buffer-independent values for the thermodynamics parameters K and 螖H associated with Cu²⁺ binding to the Cu_B site of CA to be 2 脳 10⁹ and 鈭?7.4 kcal/mol, respectively.