Influence of solvent polarity and hydrogen bonding on the EPR parameters of a nitroxide spin label studied by 9-GHz and 95-GHz EPR spectroscopy and DFT calculations

• The isotropic and anisotropic hyperfine coupling constants and g-values of the nitroxide spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (MTSSL) were determined from 9-GHz and 95-GHz electron paramagnetic resonance (EPR) measurements in various solvents with a large distribution in polarity and proticity. The parameters Aiso, giso, Azz, and gxx of MTSSL were found to be sensitive to changes in solvent properties, where A-values increased and g-values decreased due to increased solvent polarity or proticity. A linear correlation was found for the isotropic (giso, Aiso) and anisotropic (gxx, Azz) parameters, respectively. Furthermore, density functional theory (DFT) calculations of the same parameters were performed for a model spin label with the possibility to vary the dielectric constant (e) of the medium and the number of hydrogen bonds formed with the nitroxide oxygen. From a qualitative analysis of experimental and calculated results, it was possible to specify the causes of the parameter shifts in more detail. In the "apolar region" (e < 25), the sensitivity of Aiso and Azz to e is large. However, in the "polar region" (e > 25), the sensitivity to epsi, is small, and the shifts in Aiso and Azz are mainly determined by the proticity of the solvent. Methanol was found to form ~1 and water ~2 hydrogen bonds to the nitroxide on average. The DFT method determined the shifts in giso and gxx due to hydrogen bonding more accurately compared with the restricted open-shell Hartree-Fock method. The anisotropic spin label-solvent data can be used in the interpretation of rigid-limit data from spin-labeled proteins to gain further insight in local environmental properties.

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