Solution Tetrahydrobiopterin Radical vs. the Enzyme-Bound Radical: A Paramagnetic Reconciliation

Background: Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) and citrulline from L-arginine, dioxygen (O2), and nicotinamide adenine dinucleotide phosphate (NADPH) in a two-step reaction, with the enzyme-bound intermediate Nω-hydroxy-L-arginine (NHA). Previous electron paramagnetic resonance (EPR) studies of NOS reaction have shown that (6R, 1'R, 2'S)-6-(l',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (H4B) acts as a single electron donor in both steps of the reaction, resulting in the transient generation of a tetrahydropterin cation radical (H4B•+). Results: H4B•+ can also be chemically generated in strongly acidic solutions. EPR studies of chemically generated H4B•+ and similar pterin radicals date back to the 1960s. However, the reported paramagnetic parameters of H4B•+ in NOS do not seem to match the corresponding reported parameters for either H4B•+ or other pterin centered radicals chemically generated in solution. In particular, the rather isotropic hyperfine coupling of ca. 45 MHz for 1H6 of H4B•+ in NOS is at least 15 MHz larger than that of H4B•+ or any other previously studies pterin solution radical. In the work reported here, a combination of 9.5 - 9.8 GHz contentious wave (cw-) EPR, 34GHz 1H electron nuclear double resonance (ENDOR), spectral simulation and Density Functional Theory (DFT) calculations were used to investigate this seeming discrepancy. Conclusion: We demonstrated that the differences in the paramagnetic parameters of the chemically generated H4B radicals in solutions and those of the H4B radicals in NOS are consistent with the presence of two different conformers of the same cation radical in the two media.

Characterisation of the Fe(III):H+ Defect Centre in Natural Amethyst

A natural single crystal of amethyst was investigated by means of continuous-wave and pulsed Electron Paramagnetic Resonance (EPR), with the aim of structurally characterizing the substitutional S2 Fe(III):H+ centre. In this centre, Fe(III) replaces Si(IV) in the tetrahedral site, whereas H+ is coupled to Fe(III) to maintain the charge balance. The spectroscopic investigations, mainly the interpretation of the Electron Spin Echo Envelope Modulation, allowed a detailed localisation of the proton to be obtained. H+ occurs in the channels crossing the crystal parallel to the crystallographic c axis, in a largely eccentric position. The Fe(III)-H+ distance, evaluated in 2.70 Å, is found associated with a non-negligible isotropic hyperfine coupling, which can be linked to the relative stability of the S2 centre in natural amethyst.

Spin-label Order Parameter Calibrations for Slow Motion

Calibrations are given to extract orientation order parameters from pseudo-powder electron paramagnetic resonance line shapes of 14N-nitroxide spin labels undergoing slow rotational diffusion. The nitroxide z-axis is assumed parallel to the long molecular axis. Stochastic-Liouville simulations of slow-motion 9.4-GHz spectra for molecular ordering with a Maier–Saupe orientation potential reveal a linear dependence of the splittings, $$2A_{\hbox{max} }$$ 2 A max and $$2A_{\hbox{min} }$$ 2 A min , of the outer and inner peaks on order parameter $$S_{zz}$$ S z z that depends on the diffusion coefficient $$D_{{{\text{R}} \bot }}$$ D R ⊥ which characterizes fluctuations of the long molecular axis. This results in empirical expressions for order parameter and isotropic hyperfine coupling: $$S_{zz} = s_{1} \times \left( {A_{\hbox{max} } - A_{\hbox{min} } } \right) - s_{o}$$ S z z = s 1 × A max - A min - s o and $$a_{o}^{{}} = \tfrac{1}{3}\left( {f_{\hbox{max} } A_{\hbox{max} } + f_{\hbox{min} } A_{\hbox{min} } } \right) + \delta a_{o}$$ a o = 1 3 f max A max + f min A min + δ a o , respectively. Values of the calibration constants $$s_{1}$$ s 1 , $$s_{\text{o}}$$ s o , $$f_{\hbox{max} }$$ f max , $$f_{\hbox{min} }$$ f min and $$\delta a_{o}$$ δ a o are given for different values of $$D_{{{\text{R}} \bot }}$$ D R ⊥ in fast and slow motional regimes. The calibrations are relatively insensitive to anisotropy of rotational diffusion $$(D_{{{\text{R}}//}} \ge D_{{{\text{R}} \bot }} )$$ ( D R / / ≥ D R ⊥ ) , and corrections are less significant for the isotropic hyperfine coupling than for the order parameter.

Modeling EPR parameters of nitrogen containing conjugated radical cations

DFT investigation on conjugated radical cations containing14N nucleus to obtain accurate isotropic hyperfine coupling constants.