Farver O, Skov L K, van de Kamp M, Canters G W, Pecht I
Department of General Chemistry, Royal Danish School of Pharmacy, Copenhagen.
Eur J Biochem. 1992 Dec 1;210(2):399-403. doi: 10.1111/j.1432-1033.1992.tb17434.x.
An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.
先前已表明，在蓝色单铜蛋白天青蛋白中，脉冲辐射产生的半胱氨酸3-半胱氨酸26自由基离子（RSSR-）与铜（II）离子之间会发生分子内电子转移过程[法弗，O. & 佩希特，I.（1989年）《美国国家科学院院刊》86，6868 - 6972]。为了进一步研究在蛋白质基质中发生的这种长程电子转移（LRET）的性质，我们现在对两种天青蛋白进行了研究，其中氨基酸通过野生型铜绿假单胞菌天青蛋白的单点突变进行了取代。在一种突变蛋白中，靠近铜配位球的甲硫氨酸残基（Met44）被带正电荷的赖氨酸残基取代（[M44K]天青蛋白），而在第二种突变体中，另一个与铜配位位点相邻的残基（His35）被谷氨酰胺取代（[H35Q]天青蛋白）。尽管这两种取代都不在分隔电子供体和受体的微环境中，但由于它们对铜位点的氧化还原电位以及反应驱动力的影响，以及对铜位点重组能的影响，预计会影响LRET速率。先前已确定，在298 K、pH 7.0条件下，野生型铜绿假单胞菌天青蛋白中从RSSR-到铜（II）的分子内电子转移速率（ΔG° = -68.9 kJ/mol）为44 ± 7 s-1。现在发现[M44K]天青蛋白突变体（ΔG° = -75.3 kJ/mol）反应速度明显更快（在298 K、pH 7.0条件下k = 134 ± 12 s-1），而[H35Q]天青蛋白突变体（ΔG° = -65.4 kJ/mol）在实验误差范围内，表现出与野生型天青蛋白相同的比速率（k = 52 ± 11 s-1，298 K，pH 7.0）。根据这些LRET速率的温度依赖性，计算出了以下活化参数：对于[H35Q]天青蛋白和[M44K]天青蛋白，ΔH‡分别为37.9 ± 1.3 kJ/mol和47.2 ± 0.7 kJ/mol，ΔS‡分别为 -86.5 ± 5.8 J/mol·K和 -46.4 ± 4.4 J/mol·K。使用分子内电子转移的马库斯关系式和上述参数，我们确定了重组能λ和电子耦合因子β。计算值与通过键的LRET机制非常吻合。
