Mechanistic Aspects Regarding the Elimination of H2O2 from C(4a)-Hydroperoxyflavin. The Role of a Proton Shuttle Required for H2O2 Elimination.

DFT calculations presented for C(4a)-hydroperoxyflavin (C(4a)-FLHOOH) at the B3LYP/6-311+G(d,p) level suggest a new mechanism for the elimination of H2O2. The calculated activation barrier for a concerted four-centered elimination (DeltaE() = 32.86 kcal/mol) strongly suggests that in the absence of interactions with the local environment a spontaneous elimination is not feasible. A proton shuttle from the N5 hydrogen to the proximal oxygen of the OOH moiety involving three water molecules has an activation barrier that is reduced to 17.11 kcal/mol. Calculations that utilize CH3OH to model the role of a local Thr or Ser residue shows that an alcohol functionality hydrogen bonded to the N5 H-atom can catalyze the elimination of H2O2 with a free energy of activation of 21.5 kcal/mol. Interaction of amines and amide residues (CH3NH2 and CH3(C═O)NH2) with the N5 locus of C(4a)-hydroperoxyflavin markedly reduce the activation barrier for H2O2 elimination relative to the concerted pathway. Proton transfer from a COOH group (DeltaG() = 8.36 kcal/mol) or the NH2 group of a positively charged Arg model (DeltaG() = 9.99 kcal/mol) to the proximal oxygen of the OOH moiety of C(4a)-FLHOOH in the TS for H2O2 elimination strongly enhances elimination of H2O2