Van Pouderoyen G, Cigna G, Rolli G, Cutruzzolà F, Malatesta F, Silvestrini M C, Brunori M, Canters G W
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Eur J Biochem. 1997 Jul 1;247(1):322-31. doi: 10.1111/j.1432-1033.1997.00322.x.
In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.
在来自铜绿假单胞菌的天青蛋白的疏水区域，通过将Met44替换为Lys以及将Met64替换为Glu创建了一个电偶极。研究了该偶极对天青蛋白电子转移性质的影响。通过光谱表征（核磁共振、电子顺磁共振和紫外可见光谱）发现，[Lys44, Glu64]天青蛋白的铜位点和整体结构均未因这两个突变而受到干扰。在高pH值下，双突变体的活性位点出现了与[Lys44]天青蛋白类似的小扰动。在中性pH值下，双突变体的电子自交换速率常数与野生型相比降低了三个数量级。讨论了这种降低的可能原因。研究了与所提出的生理氧化还原伙伴细胞色素c551和亚硝酸还原酶的电子转移，并在马库斯框架下对数据进行了分析。通过该分析证实，天青蛋白的疏水区域是与这两个伙伴的相互作用位点，并且细胞色素c551利用其疏水区域，而亚硝酸还原酶利用带负电荷的表面积进行电子转移。
