Berry S M, Wang X, Lu Y
Department of Chemistry, The University of Illinois at Urbana-Champaign, Urbana 61801, USA.
J Inorg Biochem. 2000 Jan 15;78(1):89-95. doi: 10.1016/s0162-0134(99)00214-7.
The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X = Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn 119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn 119X (X = Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.
铜A中心是一种双核Cu2S2（半胱氨酸）电子转移中心，存在于细胞色素c氧化酶和一氧化二氮还原酶中。在之前关于赤道面组氨酸配体对铜A中心还原电位、电子转移和光谱性质影响的研究中，将工程化铜A蓝蛋白中的His120突变为天冬酰胺、天冬氨酸和丙氨酸。这些突变体相同的吸收光谱和电子顺磁共振光谱表明，一个共同的配体与铜中心结合。为了鉴定这种替代配体，构建并纯化了His120Gly铜A蓝蛋白突变体。吸收光谱和X波段电子顺磁共振光谱表明，His120Gly与其他His120X（X = 天冬酰胺、天冬氨酸、丙氨酸）突变蛋白相似。用氯离子、咪唑和叠氮化物进行滴定表明，替代配体不能与外源性配体交换。通过分析铜A蓝蛋白晶体结构，然后在His120Gly突变体中将可能的内部配体天冬酰胺119转化为天冬氨酸、丝氨酸或丙氨酸，研究了内部氨基酸作为His120X突变蛋白中His120替代配体的可能性。双突变体H120G/天冬酰胺119X（X = 天冬氨酸、丝氨酸或丙氨酸）的紫外可见吸收光谱和电子顺磁共振光谱与His120Gly和其他His120X突变体相同，表明天冬酰胺119不是His120X突变蛋白中替代His120的内部配体。这些结果证明了铜A蓝蛋白双核His120突变体具有显著的稳定性。
