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 oneelectron/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 enzymebound FADs are in reasonable agreement with experimental data. The calculations also yielded the pK a values for the one-electron reduced semiquinone (FH) and the fully reduced hydroquinone (FH 2) forms. The pK a of the FAD semiquinone in CHOX was found to be around 4, which is 4 units lower than that in the enzymefree 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 (∼5 kcal/mol) to the oxidized state. The reduction potential of lumiflavin was also computed using DFT (M06-L and B 3LYP 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 le -71H + 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.