Oxygen activation by P450cin: Protein and substrate mutagenesis

A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450_cin (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450_cin N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole. To explore further how P450_cin controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450_cin indicated that the Thr-243 was not involved in controlling the protonation of the hydroperoxy species. In addition, the N242T mutant did not enhance the rate and/or efficiency of catalytic turnover of cineole by P450_cin. In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo2.2.2]octane (cinane). An analogous experiment with P450_EryF showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450_cin with either of these alternative substrates (camphane or cinane) revealed that in the absence of the ethereal oxygen there was still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. Again the substrate itself was not found to be important in controlling oxygen activation, in contrast to P450_EryF, but was shown to be essential for regio- and stereoselective substrate oxidation. Thus, it still remains unclear how dioxygen activation in the catalytic turnover of cineole by P450_cin is controlled.