The importance of the mechanism-based inactivation (MBI) of enzymes, which has a variety of physiological effects and therapeutic implications, has been garnering appreciation. Density functional theory calculations were undertaken to gain a clear understanding of the MBI of a cytochrome P450 enzyme (CYP2B4) by tert-butylphenylacetylene (tBPA). The results of calculations suggest that, in accordance with previous proposals, the reaction proceeds via a ketene-type metabolic intermediate. Once an oxoiron(IV) porphyryn π-cation radical intermediate (compound I) of P450 is generated at the heme reaction site, ketene formation is facile, as the terminal acetylene of tBPA can form a C-O bond with the oxo unit of compound I with a relatively low reaction barrier (14.1 kcal/mol). Unexpectedly, it was found that the ketene-type intermediate was not very reactive. Its reaction with the hydroxyl group of a threonine (Thr302) to form an ester bond required a substantial barrier (38.2 kcal/mol). The high barrier disfavored the mechanism by which these species react directly. However, the introduction of a water molecule in the reaction center led to its active participation in the reaction. The water was capable of donating its proton to the tBPA molecule, while accepting the proton of threonine. This water-mediated mechanism lowered the reaction barrier for the formation of an ester bond by about 20 kcal/mol. Therefore, our study suggests that a water molecule, which can easily gain access to the threonine residue through the proton-relay channel, plays a critical role in enhancing the covalent modification of threonine by terminal acetylene compounds. Another type of MBI by acetylenes, N-alkylation of the heme prosthetic group, was less favorable than the threonine modification pathway.