The mechanisms of cytochrome P450 (CYP) catalyzed C-C bond cleavage have been strongly debated and difficult to unravel. Herein, deformylation mechanisms of the sterol 14 alpha-demethylase (CYP51) from Mycobacterium tuberculosis are elucidated using molecular dynamics simulation, density functional theory, and hybrid quantum mechanics/molecular mechanics methods. These results provide strong theoretical support for the operation of the peroxo intermediate in CYP-catalyzed deformylation. Molecular dynamics simulations support the lanosterol carboxaldehyde intermediate diverts the hydrogen-bonded network of water putatively involved in proton delivery to peroxo and compound 0 (Cmpd 0) away from the O-2 ligand. In the presence of the aldehyde substrate, the peroxo intermediate is trapped as the peroxohemiacetal without an apparent barrier, which may then be protonated in the active site. The unprotonated peroxohemiacetal provides a branch point for a concerted deformylation mechanism; however, a stepwise mechanism initiated by cleavage of the C-C bond was found to be more energetically feasible. Population analyses of the peroxoformate/deformylated substrate complex indicate that heterolytic cleavage of the C-C bond in the enzyme environment generates a carbanion at C14. Conversely, in the absence of the protein electrostatic background, the C-C cleavage reaction proceeds homolytically, indicating that the active site environment exerts a strong modulatory effect on the electronic structure of this intermediate. If the peroxohemiacetal is protonated, this species preferentially expels formic acid through an O-O cleavage transition state. After expulsion of the formyl unit, both proton-independent and -dependent pathways converge to a complex containing compound II, which readily abstracts the 15 alpha-hydrogen, thereby inserting the 14,15 double bond into the steroid skeleton. Parallel studies considering nucleophilic addition of Cmpd 0 to the aldehyde intermediate indicated that this reaction proceeds with high energetic barriers. Finally, the hydrogen atom abstraction and proton coupled electron transfer mechanism (J. Am. Chem. Soc. 2005, 127, 5224-5237) for compound I (Cmpd I) mediated deformylation of the geminal diol was considered in the context of the protein environment. In contrast to gas phase calculations, triradicaloid and pentaradicaloid Cmpd I states failed to initiate a concerted deformylation of the geminal diol. This study provides a unified mechanistic view consistent with decades of experiments aimed at understanding the deformylation reaction. Additionally, these results provide general mechanistic insight into the catalytic mechanisms of several biosynthetic and xenobiotic-oxidizing CYP enzymes of biomedical importance.