Influence of Protein Environment on the Electron Paramagnetic Resonance Properties of Flavoprotein Radicals: A QM/MM Study

Abstract

The neutral and anionic semiquinone radicals of the flavin adenine dinucleotide (FAD) cofactor noncovalently bound in glucose oxidase from A. niger are examined with the aid of QM/MM molecular modeling methods, enabling complete inclusion of the protein environment. Recently, the electron paramagnetic resonance (EPR) characteristics, the anisotropic g tensor and all the significant hyperfine couplings, of these flavoprotein radicals were determined at high resolution (J. Phys. Chem. B 2008, 112, 3568). A striking difference between the neutral and anionic radical forms was found to be a shift in the g(y) principal value. Within the QM/MM framework, geometry optimization and molecular dynamics simulations are combined with EPR property calculations, employing a recent implementation by some of the authors in the CP2K software package. In this way, spectroscopic characteristics are computed on the fly during the MD simulations of the solvated protein structure, mimicking as best as possible the experimental conditions. The general agreement between calculated and experimental EPR properties is satisfactory and on par with those calculated with other codes (Gaussian 03, ORCA). The protonation state of two histidines (His559 and His516) at the catalytic site of this flavoprotein is found to have a remarkable influence on the isotropic hyperfine coupling of one of the methyl groups on the neutral FAD radical, which is consistent with experimental findings in other flavoproteins (J. Biol. Chem. 2007, 282, 4738). Furthermore, the shift in the g(y) principal values between the neutral and anionic radicals is well reproduced by QM/MM simulations. Incorporation of at least the nearest protein environment of the cofactor radicals proves to be vital for a correct reproduction, indicating that this shift is a global feature of the protein rather than a local one. In addition, QM/MM techniques are used to make a prediction of relative angles between important spectroscopic principal directions, which are not readily determined by conventional EPR experiments. Significantly, the directions of the g(x) and the g(y) components of the g-tensor that lie in the plane of the isoalloxazine moiety are rotated by approximately 59° between the neutral and the anionic radicals.

Inhomogeneous ensembles of radical pairs in chemical compasses

“…due to substrate binding, altered geometries of hydrogen bonds, surrounding amino acids have been shown experimentally535455565758596061 and theoretically436263. If such inherent heterogeneities existed, they could potentially lead to significantly larger levels of variation than the 2% level assumed here.…”

“…Secondly, through QM-MM simulations hyperfine interactions of flavin radicals were estimated to fluctuate on short time scales (picoseconds) with an average magnitude of 0.06 mT43. The smallest magnitude was 0.02 mT while the largest 0.1 mT.…”

“…We consider the hyperfine tensor components taken from flavin and tryptophan radicals30 as average values of Gaussian distributions with fixed-width of 0.06 mT. We choose a fixed-width of 0.06 G because this is the average size of variations of many hyperfine tensors in a flavin radical available from the literature43. For each hyperfine tensor component we generate 3000 samples of normally distributed values, and then compute 3000 singlet yields of each eight-nuclei radical pair system.…”

“…in photosynthetic proteins42. Fluctuations of hyperfine interactions in a flavin cofactor were estimated on short time scale43, and similarly sized variations are expected to occur on larger time scale as well. Here, we assume a relatively modest level of variation, i.e.…”

Four-component relativistic density functional theory with the polarisable continuum model: application to EPR parameters and paramagnetic NMR shifts

“…However, the solute is treated quantum mechanically whereas the solvent molecules are treated with a classical force field [54]. Although the QM/MM methods have received considerable popularity and several works have recently been devoted to QM/MM modeling of electronic g-tensors and hyperfine coupling constants of paramagnetic species in solution, [56][57][58][59][60][61] various questions related to the number of snapshots required to reach convergence of di↵erent properties or the size of the solvation shells remain di cult to answer. These aspects have limited the use of the QM/MM methodology as a widespread black-box computational protocol.…”