Combined Quantum Mechanical and Molecular Mechanical Simulations of One- and Two-Electron Reduction Potentials of Flavin Cofactor in Water, Medium-Chain Acyl-CoA Dehydrogenase, and Cholesterol Oxidase

Abstract

Flavin adenine dinucleotide (FAD) is a common cofactor in redox proteins, and its reduction potentials are controlled by the protein environment. This regulation is mainly responsible for the versatile catalytic functions of flavoenzymes. In this article, we report computations of the reduction potentials of FAD in medium-chain acyl-CoA dehydrogenase (MCAD) and cholesterol oxidase (CHOX). In addition, the reduction potentials of lumiflavin in aqueous solution have also been computed. Using molecular dynamics and free-energy perturbation techniques, we obtained the free-energy changes for two-electron/two-proton as well as one-electron/one-proton addition steps. We employed a combined quantum mechanical and molecular mechanical (QM/MM) potential, in which the flavin ring was represented by the self-consistent-charge density functional tight-binding (SCC-DFTB) method, while the rest of the enzyme-solvent system was treated by classical force fields. The computed two-electron/two-proton reduction potentials for lumiflavin and the two enzyme-bound FADs are in reasonable agreement with experimental data. The calculations also yielded the pKa values for the one-electron reduced semiquinone (FH*) and the fully reduced hydroquinone (FH2) forms. The pKa of the FAD semiquinone in CHOX was found to be around 4, which is 4 units lower than that in the enzyme-free state and 2 units lower than that in MCAD; this supports the notion that oxidases have a greater ability than dehydrogenases to stabilize anionic semiquinones. In MCAD, the flavin ring interacts with four hydrophobic residues and has a significantly bent structure, even in the oxidized state. The present study shows that this bending of the flavin imparts a significant destabilization (approximately 5 kcal/mol) to the oxidized state. The reduction potential of lumiflavin was also computed using DFT (M06-L and B3LYP functionals with 6-31+G(d,p) basis set) with the SM6 continuum solvation model, and the results are in good agreement with results from explicit free-energy simulations, which supports the conclusion that the SCC-DFTB/MM computation is reasonably accurate for both 1e(-)/1H+ and 2e(-)/2H+ reduction processes. These results suggest that the first coupled electron-proton addition is stepwise for both the free and the two enzyme-bound flavins. In contrast, the second coupled electron-proton addition is also stepwise for the free flavin but is likely to be concerted when the flavin is bound to either the dehydrogenase or the oxidase enzyme.

Formate Oxidase, an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family, Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

“…3,4,40) The bending in the oxidase and dehydrogenase destabilize their oxidized states and the bent angle has been shown to be a key indicator of the geometric effects of the enzyme matrix on the ORP. 40) The bending of the isoalloxazine ring of 8-formyl-FAD in FOD AO (Fig. 5A) must affect the ORP of 8-formyl-FAD in the enzyme.…”

Toward robust computational electrochemical predicting the environmental fate of organic pollutants

“…A large number of publications achieved virtually experimental accuracy of quantum-chemical calculations of reduction potential E 0 . [3][4][5][6][7][8][9][10][11][12][13][14][15][16] However, it is still not clear whether the experimental accuracy is reached due to an accurate quantum-chemical prediction of both DG EA gas and DDG solv or the calculated values of E 0 are just accurate due to the cancelation of errors. In addition, those studies which report experimental accuracy for different classes of organic compounds use different levels of theory to account for electron correlation (sometimes extremely time consuming) and different basis sets.…”

Calculating solution redox free energies with ab initio quantum mechanical/molecular mechanical minimum free energy path method

“…[33][34][35] For large biomolecular systems, the computational cost for the QM calculation of the active site and phase space sampling of the protein severely limit the application of the direct QM/MM MD simulations. Although the semiempirical QM methods, including the empirical valence bond model 36 and the self-consistentcharge density functional tight-binding method, 37 can largely reduce the computational cost in the QM evaluations, [38][39][40][41] the reliability and transferability of the semiempirical QM methods still limited their applications.…”