Hay M, Richards J H, Lu Y
Department of Chemistry, University of Illinois, Urbana 61801, USA.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):461-4. doi: 10.1073/pnas.93.1.461.
A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.
一种紫色铜中心的蛋白质类似物已通过重组蓝铜蛋白（铜绿假单胞菌天青蛋白）构建而成，即将包含与蓝铜中心的三个配体的环替换为来自反硝化副球菌的细胞色素c氧化酶（COX）中CuA中心的相应环。该类似物在紫外和可见光区域的电子吸收（紫外-可见）光谱以及电子顺磁共振（EPR）光谱与来自反硝化副球菌的COX中的天然CuA中心的光谱非常相似。在添加Cu2+和Cu+的混合物后可获得上述光谱。仅添加Cu2+会产生一个紫外-可见光谱，该光谱由紫色铜中心和蓝铜中心的吸收组成。通过在空气中长时间孵育或添加过量抗坏血酸，该光谱可转变为纯紫色铜的光谱。此处报道的天青蛋白突变体是一种工程化紫色铜中心的实例，其A480/A530比值大于1且无可检测到的超精细结构，类似于牛心COX和反硝化副球菌中CuA位点的情况。
