Electronic structure studies of flavin radicals

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One of the most fundamental questions in biochemistry and biophysics is, how cofactor specificity is achieved for a particular catalytic reaction via the modulation of the coenzyme by the apoprotein. To this end, the unpaired electron-spin density is an important physical quantity to probe frontier orbitals of aromatic cofactors in their radical state. By such an approach, the contribution of electronic structure may be evaluated. Hyperfine coupling constants as observed by electron paramagnetic resonance (EPR) and thereupon-based methods, such as electron-nuclear double resonance (ENDOR) or hyperfine sublevel correlation (HYSCORE) spectroscopy, are determined by the electron-spin densities at or near the positions of magnetic nuclei. In this project we strive for an as complete as possible hyperfine mapping of the atoms directly composing the pi-ring system of the ubiquitously encountered flavin cofactor, specifically, of the carbons and the nitrogens. Previous reports in the literature mostly relied on presenting hyperfine couplings of protons bound to the 7,8-dimethyl isoalloxazine ring to draw, rather indirect, conclusions on the electronic structure of paramagnetic flavins. Using a library of selectively 13C- and 15N-labeled flavin coenzyme isotopologs, we have applied pulsed EPR methods to directly map the hyperfine structure of the pi-ring system of flavin semiquinones in various proteins (photolyase, LOV2 domain of phototropin, flavodoxin) that stabilize the neutral radical form. In the project extension, we will strive for sophisticated theoretical analyses of the hyperfine mappings using hybrid quantum-mechanical/molecular-mechanical (QM/MM) computations. We expect from the results to predict, which specific protein-cofactor interactions are critical for the function-specific distribution of unpaired electron-spin density that is reflected in the size and anisotropy of hyperfine couplings.