Mechanism of oxidation of pi bonds by cytochrome P-450. Electronic requirements of the transition state in the turnover of phenylacetylenes.

Substituted phenylacetylenes are oxidized by purified reconstituted rat liver cytochrome P-450b to the corresponding 2-arylacetic acids. A linear free energy correlation with a rho value of -2.2 exists between the rates of formation of the arylacetic acids and the substituent sigma constants. The enzyme is simultaneously inactivated but the inactivation rates, in contrast to those of metabolite formation, are essentially identical for all the phenylacetylenes. Inactivation is thus insensitive to the electronic nature of the substituents. The metabolite: inactivation ratio consequently decreases from 38 for p-methylphenylacetylene to 4 for p-nitrophenylacetylene. Replacement of the terminal proton of phenylacetylene with deuterium gives rise to an isotope effect of 1.7 on metabolite formation without altering enzyme inactivation. The differential effect of substituents on heme alkylation and metabolite formation requires the two pathways to diverge early in the catalytic process and argues against mechanisms that involve radical cation intermediates.