The P450eryF enzyme (CYP107A1) hydroxylates 6-deoxyerythronolide B to erythronolide B during erythromycin synthesis by Saccharopolyspora erythraea. In many P450 enzymes, a conserved "acid-alcohol pair" is believed to participate in the proton shuttling pathway for O-2 activation that generates the reactive oxidant (Compound I, Cpd I). In CYPI07A1, the alcohol-containing amino acid is replaced with alanine. The crystal structure of DEB bound to CYP107A1 indicates that one of the substrate hydroxyl groups (5-OH) may facilitate proton transfer during O-2 activation. We applied molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) techniques to investigate substrate-mediated O-2 activation in CYP107A1. In the QM/MM calculations, the QM region was treated by density functional theory, and the MM region was represented by the CHARMM force field. The MD simulations suggest the existence of two water networks around the active site, the one found in the crystal structure involving E360 and an alternative one involving E244. According to the QM/MM calculations, the first proton transfer that converts the peroxo to the hydroperoxo intermediate (Compound 0, Cpd 0) proceeds via the E244 water network with direct involvement of the 5-OH group of the substrate. For the second proton transfer from Cpd 0 to Cpd I, the computed barriers for the rate-limiting homolytic O-O cleavage are similar for the E360 and E244 pathways, and hence both glutamate residues may serve as proton source in this step.