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

Abstract

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 A iso , g iso , A zz , and g xx 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 (g iso , A iso ) and anisotropic (g xx , A zz ) 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 ( ) 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" ( < 25), the sensitivity of A iso and A zz to is large. However, in the "polar region" ( > 25), the sensitivity to is small, and the shifts in A iso and A zz 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 g iso and g xx 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.

Heterogeneity in the Nitroxide Micro-Environment: Polarity and Proticity Effects in Spin-Labeled Proteins Studied by Multi-Frequency EPR

“…Starting from the fact that the analysis of the fraction plot indicates that the g xx 0 component is prevalently populated in apolar environments, while the g xx 2 component describes a fraction of population energetically favored in polar environments, one might surmise that g xx 0 corresponds to a non-Hbonded nitroxide moiety and g xx 2 to a H-bonded moiety, mostly favored in polar environments, which are usually also characterized by a higher amount of bulk water molecules. If this was the case, the difference between the average values of g xx 0 and g xx 2 of almost 900 ppm would not be in line with both the theoretical Dg xx value of &400 ppm, as calculated for the addition of one H-bond [2,8], and the experimental Dg xx value of 600 ppm, as found in the case of the spin-labeled 5-doxyl stearic acid in lipid membranes [6]. The Dg xx of 2100 ppm calculated by Engström et al [11] for the addition of one H-bond is admittedly exceeding the experimentally observed values by far.…”

“…In fact, for u = 20°, a slope of 1.1 9 10 -3 mT -1 was calculated. Furthermore, a slope of 0.8 9 10 -3 mT -1 was obtained from measurements of unbound MTSSL in different solvents [8].…”

“…To reduce the number of fit parameters, the line width broadening function was determined from the g yy and g zz spectral regions. The homogeneous line width was determined from the g zz region, since it is constant in good approximation [8], while the inhomogeneous line width was taken from the g yy region. The A xx and A yy components of the hyperfine coupling tensor, which are less sensitive to polarity changes, were approximated as 0.52 and 0.45 mT for 14 N-isotope spin-label side chains, and 0.72 and 0.64 mT for 15 N-isotope spin-label side chains.…”

“…For nitroxide labels, the g anisotropy is already resolved at W-band EPR (95 GHz/3.4 T) and can be further exploited at higher frequency/field settings (e.g., 275 GHz/9.8 T and 360 GHz/12.9 T). Theoretical studies have been performed by several groups to describe the effect of polarity and propensity for H-bond formation on the A-and g-tensor elements at different levels of sophistication and with different focuses [2,[8][9][10][11].…”

Investigation of contrasting hydrogen bonding pattern of 3-(phenylamino)-cyclohexen-1-one with solvents in the ground and excited states

“…The modes of interactions also vary if the electronic structure of the molecule in the ground and excited states are very different. Many experimental and quantum chemical investigations are found in literature reporting the studies on role of media in the electronic structure and properties of different organic molecules [13][14][15][16][17].…”