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Structural Investigation of a High‐Affinity Mn II Binding Site in the Hammerhead Ribozyme by EPR Spectroscopy and DFT Calculations. Effects of Neomycin B on Metal‐Ion Binding
Author(s) -
Schiemann Olav,
Fritscher Jörg,
Kisseleva Natalja,
Sigurdsson Snorri Th.,
Prisner Thomas F.
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200300653
Subject(s) - chemistry , electron paramagnetic resonance , hammerhead ribozyme , ribozyme , binding site , spectroscopy , binding energy , metal , nuclear magnetic resonance spectroscopy , crystallography , ion , stereochemistry , nuclear magnetic resonance , rna , biochemistry , physics , gene , organic chemistry , quantum mechanics , nuclear physics
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 K d of 4.4 μ M 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 κ of 0.7 MHz, an asymmetry parameter η of 0.4, and an isotropic hyperfine coupling constant of A iso ( 14 N)=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 κ( 14 N)=+0.80 MHz , η=0.324, and A iso ( 14 N)=+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 ( K d =1.2 μ M ) 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).