Effect of Aromatic Interactions on Flavin's Redox Potential: A Theoretical Study Michael A. North and Sudeep Bhattacharyya Department of Chemistry, University of Wisconsin-Eau Claire, WI Abstract Flavins, substituted isoalloxazines, are redox active cofactors ubiquitous among oxidoreductases that catalyze diverse chemical reactions. They also exhibit photoinduced electron transfers and have great potentials as sensors and as light-harvesting chromophores. Versatility in chemical functions in flavin containing enzymes arise mainly due their unique ability to undergo both one- and two-electron reductions. Modulation of the redox potential is accomplished by various hydrogen bonding interactions with flavin ring atoms, as well as  -stacking interactions with aromatic sidechains. Although, recent studies revealed the role of hydrogen bonding and electrostatics on the charge separation upon reduction (1), the significance of aromatic functionalities and their impact on flavin's redox potential have remained poorly understood. In the present study, these noncovalent interactions, in various flavin-aromatics, have been studied and their impact on the electrochemical properties investigated. Using density functional theory (DFT), the free energy changes of these flavin-aromatic non-covalent hybrids have been calculated in their gas- phase and condensed-phase. The geometry and energetics of these molecular hybrids derived from this study will be presented. Background Theory and MethodsResults Conclusions Objective  Gas-phase free energy changes were obtained using electronic structure calculations.  M05-2X, M06-2X, and B3LYP functionals with 6-31G+ (d,p) basis set were used. M05-2X and M06-2X are parameterized to include dispersion interactions and provide “medium-range” (< 5 Å) electron correlation (2)  Solvation free energy changes were calculated by using polarizable continuum model (PCM)  Gas-phase vibrational-rotational free energies at 298 K, were calculated with the harmonic oscillator-rigid rotor approximation  The electronic free energy was approximated as –RT lnd, where R is the gas constant, T is the temperature, and d is the electronic degeneracy of the ground state. Its contribution is < 0.5 kcal/mol and was neglected (3) Flavin’s 2e ─ /H + Reduction Process F is a shorthand representation of flavin,  Modeling the geometry of flavin-bound aromatics  Effect of  -  interactions on the redox potentials of flavin  Effect of substitutions on the flavin-bound aromatics  Modeling the geometry of flavin-bound aromatics  Effect of - - interactions on the redox potentials of flavin  Effect of substitutions on the flavin-bound aromatics ( ) (1)Rauschnot, J. C.; Yang, C.; Yang, V.; Bhattacharyya, S. J. Phys. Chem. B 2009, 113, (2)Zhao, Y. and Truhlar, D. G. Acc. Chem. Res. 2009, 41, (3)Bhattacharyya, S; Stankovich, M. T.; Truhlar, D. F.; Gao, J J. Phys. Chem. A 2007, 111, References- Acknowledgements- -Office of Research Sponsored Programs at University of Wisconsin Eau Claire. - Learning and Technology Services at University of Wisconsin Eau Claire. -This research was supported in part by the National Science Foundation through TeraGrid resources provided by National Center for Supercomputing Applications (NCSA) and Louisiana Optical Network Initiative (LONI) Queen Bee system under grant number TG- DMR We specifically acknowledge the assistance of Dr. Sudhakar Pamidighantam.  The electronic structure calculations were done using Kohn-Sham DFT scheme, where the total energy of a multi-electronic system is expressed as a functional of the electron density,  (r) E[  (r)] = E KE [  (r)] + E elec-elec [  (r)] +E XC [  (r)] + E nuc-elec [  (r)]  Born-Oppenheimer Approximation - This assumes that the electrons follow the motion of nucleii instantaneously – i.e. the distribution of electrons remain stationary with respect to a particular nucleus while it moves. This allows to separate electronic motion from nuclear motion expressing the energy as a function of nuclear coordinates.  Rigid Rotor Harmonic Oscillator Approximation - This assumes that the vibrational and rotational energies act independently of one another – i.e., that the rotations occur so fast as the molecule vibrates that the inter- atomic bond distance remains a constant, as far as the rotational energy is concerned. As a result, the energies are additive – i.e., the total internal energy is written as a sum of the energies. Gas-phase Free EnergiesSolvation Free Energies Aqueous-phase Free Energies Flavin and Substituted Benzene  Structures of the flavin-bound benzene and flavin-bound substituted benzene demonstrate that the π - π stacking interactions are modeled with adequate accuracy.  The benzene ring interacts with the uracil moiety of the flavin ring.  Computed free energy for the 2e – /H + reduction of flavin shows little difference between M05-2x and M06-2x.  Substitutions on various positions of the benzene ring produce significant changes (3-5 kcal/mol) in the energetics.  Changes due to the substitution effects of an electron donating (NH 2 -) and electron withdrawing group (Cl-) on the energetics were found to be within 3 kcal/mol.  Computations also show that M05-2x and M06-2x produce significantly different energetics from M06L and B3LYP. Model other substitutions and study their effect. Try different substitutions on the rings, to observe the effect of different groups have on flavin’s redox potential. Model the binding free energy of these non-covalent molecular complexes. Future Directions Free Energy Calculations