Schiemann Olav, Fritscher Jörg, Kisseleva Natalja, Sigurdsson Snorri Th, Prisner Thomas F
Department of Physical and Theoretical Chemistry, and Center for Biological Magnetic Resonance, Johann Wolfgang Goethe University, Marie-Curie-Strasse 11, 60439 Frankfurt/Main, Germany.
Chembiochem. 2003 Oct 6;4(10):1057-65. doi: 10.1002/cbic.200300653.
Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity Mn(II) binding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant Ke of 4.4 microM in buffer solutions containing 1 M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant kappa of 0.7 MHz, an asymmetry parameter eta of 0.4, and an isotropic hyperfine coupling constant of Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the Mn(II) ligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by Mn(II) in crystals (MacKay et al., Nature 1994, 372, 68 and Scott et al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the Mn(II) site in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo-II basis set for all other atoms yielded values of kappa(14N)=+0.80 MHz, eta=0.324, and Aiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that Mn(II) is released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 microM) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11 186).
利用电子顺磁共振光谱和密度泛函理论方法研究了锤头状核酶中单个高亲和力锰（II）结合位点的结构。如连续波（cw）电子顺磁共振监测的滴定所示，在含有1 M氯化钠的缓冲溶液中，该结合位点的解离常数Ke为4.4 μM。电子自旋回波包络调制（ESEEM）和超精细能级相关（HYSCORE）实验相结合表明，该结合位点中的顺磁性锰（II）离子与单个氮原子耦合，其四极耦合常数κ为0.7 MHz，不对称参数η为0.4，各向同性超精细耦合常数Aiso(14N)=2.3 MHz。所有这三个电子顺磁共振参数都对锰（II）配体球的排列敏感，因此可用于确定结合位点的结构。该结合位点的一个可能位置可能在晶体中发现被锰（II）占据的G10.1、A9位点（麦凯等人，《自然》1994年，372卷，68页；斯科特等人，《科学》1996年，274卷，2065页）。为了确定结合位点在冷冻溶液中的结构是否相同，我们基于晶体中锰（II）位点的结构对电子顺磁共振参数进行了密度泛函理论计算。使用BHPW91密度函数结合锰（II）中心的9s7p4d基组和所有其他原子的Iglo-II基组进行计算，得到κ(14N)=+0.80 MHz、η=0.324和Aiso(14N)=+2.7 MHz的值，与实验获得的电子顺磁共振参数非常吻合，这表明在晶体和冷冻溶液中发现的结合位点是相同的。此外，我们通过电子顺磁共振证明，当氨基糖苷类抗生素新霉素B（Kd=1.2 μM）与锤头状核酶结合时，锰（II）从该位点释放。新霉素B先前已被证明可抑制该核酶的催化活性（乌伦贝克等人，《生物化学》1995年，34卷，11186页）。
