Structural Investigation of a High‐Affinity MnIIBinding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations. Effects of Neomycin B on Metal‐Ion Binding

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Abstract
Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high‐affinity MnIIbinding site in the hammerhead ribozyme. This binding site exhibits a dissociation constantKdof 4.4 μMin buffer solutions containing 1 MNaCl, 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 constantκof 0.7 MHz, an asymmetry parameterηof 0.4, and an isotropic hyperfine coupling constant ofAiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the MnIIligand 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 MnIIin crystals (MacKay et al., Nature1994, 372,68 and Scott et al., Science1996, 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 MnIIsite 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 ofκ(14N)=+0.80 MHz,η=0.324, andAiso(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 MnIIis released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 μM) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry1995, 34,11 186).