Neri F, Kok D, Miller M A, Smulevich G
Dipartimento di Chimica, Universita' di Firenze, Via G. Capponi 9, 50121 Firenze, Italy.
Biochemistry. 1997 Jul 22;36(29):8947-53. doi: 10.1021/bi970248+.
The electronic absorption and resonance Raman spectra of the fluoride complexes of various peroxidases and selected site-directed mutants have been studied at pH 5.0, and compared to the spectra obtained for the myoglobin-F adduct. It is shown that the electronic absorption maxima depend on the degree of conjugation between the porphyrin macrocycle and the vinyl substituents. Moreover, it is confirmed that the wavelength of the CT1 band is a sensitive probe of axial ligand polarity and of its interaction with the distal protein residues. The results highlight the different mechanism of stabilization of the fluoride ligand exerted by the distal residues in myoglobin and peroxidases. In peroxidases, the Arg is determinant in controlling the ligand binding via a strong hydrogen bond between the positively charged guanidinium group and the anion. Mutation of Arg to Leu decreases the stability of the complex by 900-fold, suggesting that this interaction stabilizes the complex by 4 kcal/mol. The distal His also contributes to the stability of the fluoride complex, presumably by accepting a proton from HF and hydrogen-bonding, through a water molecule, to the anion. Mutation of His to Leu decreases the stability of the fluoride complex by 30-fold, suggesting that this interaction is much weaker than the interaction with the distal Arg. For Mb, the distal His is solely responsible for stabilization of the exogenous ligand.
在pH 5.0条件下研究了各种过氧化物酶及其选定的定点突变体的氟化物配合物的电子吸收光谱和共振拉曼光谱，并与肌红蛋白-F加合物的光谱进行了比较。结果表明，电子吸收最大值取决于卟啉大环与乙烯基取代基之间的共轭程度。此外，证实CT1带的波长是轴向配体极性及其与远端蛋白质残基相互作用的灵敏探针。结果突出了肌红蛋白和过氧化物酶中远端残基对氟化物配体的不同稳定机制。在过氧化物酶中，精氨酸通过带正电荷的胍基与阴离子之间的强氢键来控制配体结合，起决定性作用。将精氨酸突变为亮氨酸会使配合物的稳定性降低900倍，这表明这种相互作用使配合物稳定了4千卡/摩尔。远端组氨酸也有助于氟化物配合物的稳定性，大概是通过从HF接受一个质子并通过水分子与阴离子形成氢键来实现的。将组氨酸突变为亮氨酸会使氟化物配合物的稳定性降低30倍，这表明这种相互作用比与远端精氨酸的相互作用弱得多。对于肌红蛋白，远端组氨酸是外源配体稳定的唯一原因。
