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首页> 外文期刊>Biochemistry >Introduction of a pi-pi Interaction at the Active Site of a Cupredoxin: Characterization of the Met16Phe Pseudoazurin Mutant.
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Introduction of a pi-pi Interaction at the Active Site of a Cupredoxin: Characterization of the Met16Phe Pseudoazurin Mutant.

机译：在铜氧还蛋白的活性位点引入pi-pi相互作用：Met16Phe伪天青素突变体的表征。

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摘要

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 weakeraxial 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.
机译：已经鉴定了1型铜蛋白假天青素（PACu）的Met16Phe突变体，其中在靠近His81配体的咪唑部分附近引入了苯环。 NMR研究表明，引入的苯环与His81的咪唑基平行。该突变对PACu（II）可见光谱中的两个S（Cys）-> Cu（II）配体-金属电荷转移带的位置有微妙的影响，并且对其强度产生更大的影响，从而导致Met16Phe的A（459）/ A（598）比为0.31，而野生型PACu（II）在pH 8时的A（453）/ A（594）比为0.43。 Met16Phe变体比野生型蛋白具有更多的轴向，该突变体的共振拉曼光谱表现出细微的差异。与它在野生型蛋白中的位置相比，Met86的AC（γH）质子在Met16Phe PACu（II）的顺磁（1）H NMR光谱中显示出更小的超精细位移，这表明其弱的轴向Cu-S（Met86 ）在突变体中的相互作用。 Met16Phe突变导致PACu还原电位增加约60 mV。配体His81的pK（a）值从野生型PACu（I）中的4.9降低到Met16Phe PACu（I）中的4.5，表明与Phe16的pi-pi接触稳定了Cu-N（His81）相互作用。 PACu的Met16Phe变体在pH 7.6（25摄氏度）下的自交换速率常数为9.8 x 10（3）M（-）（1）s（-）（1），相比之下其值小得多（3.7）在相同条件下，野生型蛋白的x 10（3）M（-）（1）s（-）（1）。 Met16Phe PACu的增强的电子转移反应性是重组能量较低的结果，这归因于His81与引入的苯环之间的pi-pi相互作用引起的额外活性位点刚性。

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