Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant.
Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant.
复制标题
在铜氧还蛋白活性位点引入 pi-pi 相互作用：Met16Phe 假天青蛋白突变体的表征。
作者：
S. Yanagisawa;Katsuko Sato;M. Kikuchi;T. Kohzuma;C. Dennison
Met 16 Phe突变的1型铜蛋白pseudoazurin（PACu），其中苯环被引入接近咪唑部分的His 81配体，已被表征。NMR研究表明，引入的苯环是平行的咪唑基团的His 81。该突变对PACu（II）可见光谱中两个S（Cys）->Cu（II）配体-金属电荷转移带的位置具有微妙的影响，并且对它们的强度具有更显著的影响，导致在pH 8下Met 16 Phe的A（459）/A（598）比为0.31，而野生型PACu（II）的A（453）/A（594）比为0.43。Met 16 Phe变异体的电子顺磁共振光谱比野生型蛋白质的电子顺磁共振光谱更轴向，并且突变体的共振拉曼光谱表现出细微的差异。Met 86的C（γ）H质子在Met 16 Phe PACu（II）的顺磁（1）H NMR谱中与其在野生型蛋白中的位置相比表现出小得多的超精细位移，这表明突变体中较弱的轴向Cu-S（Met 86）相互作用。Met 16 Phe突变导致PACu的还原电位增加约60 mV。配体His 81的pK（α）值从野生型PACu（I）中的4.9降低到Met 16 Phe PACu（I）中的4.5，表明与Phe 16的pi-pi接触稳定了Cu-N（His 81）相互作用。PACu的Met 16 Phe变体在pH 7.6（25 ℃）下的自交换速率常数为9.8 × 10（3）M（-）（1）s（-）（1），而野生型蛋白在相同条件下的自交换速率常数为3.7 × 10（3）M（-）（1）s（-）（1）。Met 16 Phe PACu的增强的电子转移反应性是由于His 81和引入的苯环之间的pi-pi相互作用引起的额外活性位点刚性导致的较低重组能的结果。
The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.