Nettles Whitnee L, Song He, Farquhar Erik R, Fitzkee Nicholas C, Emerson Joseph P
†Department of Chemistry, Mississippi State University, Box 9573, Mississippi State, Mississippi 39762-9573, United States.
‡National Synchrotron Light Source, Brookhaven National Laboratory, Case Western Reserve University Center for Synchrotron Biosciences, Upton, New York 11973, United States.
Inorg Chem. 2015 Jun 15;54(12):5671-80. doi: 10.1021/acs.inorgchem.5b00057. Epub 2015 May 26.
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 Cu(2+) ions bind independently forming two noncoupled type II copper centers in CA (CuA and CuB). However, the location and coordination mode of the CuA site in solution is unclear, compared to the CuB site that has been well-characterized. Using paramagnetic NMR techniques and X-ray absorption spectroscopy we identified an N-terminal Cu(2+) binding location and collected information on the coordination mode of the CuA site in CA, which is consistent with a four- to five-coordinate N-terminal Cu(2+) binding site reminiscent to a number of N-terminal copper(II) binding sites including the copper(II)-amino terminal Cu(2+) and Ni(2+) and copper(II)-β-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 Cu(2+) binding data to the high-affinity CuA site, we derived pH- and buffer-independent values for the thermodynamics parameters K and ΔH associated with Cu(2+) binding to the CuB site of CA to be 2 × 10(9) and -17.4 kcal/mol, respectively.
人类碳酸酐酶（CA）是一种经过充分研究的、稳定的含单核锌金属蛋白，它是研究水溶液中与金属离子配位化学相关的热力学的优秀生物配体系统。人碳酸酐酶II（CA）的脱辅基形式以高亲和力结合2当量的铜（II）。Cu(2+)离子独立结合，在CA中形成两个非耦合的II型铜中心（CuA和CuB）。然而，与已得到充分表征的CuB位点相比，溶液中CuA位点的位置和配位模式尚不清楚。利用顺磁核磁共振技术和X射线吸收光谱，我们确定了一个N端Cu(2+)结合位置，并收集了关于CA中CuA位点配位模式的信息，这与一个四到五配位的N端Cu(2+)结合位点一致，类似于许多N端铜（II）结合位点，包括铜（II）-氨基末端Cu(2+)和Ni(2+)以及铜（II）-β-淀粉样蛋白复合物。此外，我们报告了对与铜（II）结合到CA相关的热力学的更详细分析。尽管我们仍然无法完全解卷积Cu(2+)与高亲和力CuA位点的结合数据，但我们得出了与Cu(2+)结合到CA的CuB位点相关的热力学参数K和ΔH的与pH和缓冲液无关的值，分别为2×10(9)和-17.4 kcal/mol。
