The recently determined crystal structure of cytochrome P450eryF (6-deoxyerythronolide B hydroxylase; CYP107Al) in its ferric heme substrate-bound form has been used to address one of the most fundamental unresolved aspects of the mechanism of oxidation common to this ubiquitous family of metabolizing heme proteins, the pathway from the twice reduced dioxygen species to the putative catalytically active ferryl oxygen species. Both of these species are too transient to have been characterized experimentally, and the transformation from one to the other has been only partially characterized. The observed requirement of two protons and the formation of water in this transformation suggests a proton-assisted dioxygen bond cleavage as a plausible pathway. However, this pathway is difficult to establish by experiment alone, and the source of the protons in the largely hydrophobic binding pocket of the P350s remains unclear. In this work we have performed molecular dynamics simulations of the twice reduced dioxygen substrate-bound form of this isozyme in order to (i)determine the plausibility of the proposed pathway to compound I formation, a proton-assisted cleavage of the dioxygen bond, and (ii) investigate the possible source of these protons. The analysis of the molecular dynamics trajectories of this species does indeed provide further evidence for this pathway and points to a source of protons. Specifically, two dynamically stable hydrogen bonds to the distal oxygen atom of the dioxygen ligand, one by the substrate and the other by a bound water, are found, consistent with the proposed proton-assisted cleavage of the bond and formation of water. In addition, an extensive dynamically stable hydrogen bond network is formed that connects the distal oxygen to Glu 360, a well-conserved residue in a channel accessible to solvent that could be the ultimate source of protons. The simulations were done for both a protonated and unprotonated Glu and led to a proposed mechanism of proton transfer by it to the distal oxygen atom. In order to validate the procedures used for the simulation of this transient twice-reduced species, we have used these same procedures to perform molecular dynamics simulations of two other forms of P450eryF, the ferric and ferryl substrate-bound species, and compared the results with experiment. The results for the ferric substrate-bound species were assessed by comparisons to the experimentally determined X-ray structure and fluctuations, and good agreement was found. The simulations performed for the ferryl substrate-bound species led to the correct prediction of the observed regio- and stereospecific hydroxylation of its natural substrate, 6-deoxyerythronolide B (6-DEB) at the 6S position. The results of these two additional studies lend credibility to the important mechanistic inferences from the simulations of the transient twice reduced dioxygen species : further evidence for a proton-assisted pathway from it to the catalytically active ferryl species and a possible source of the protons.