Oxygen activation by P450 cin: 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. P450cin (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 P450cin 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 P450cin controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450cin 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 P450cin. In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo[2.2.2]octane (cinane). An analogous experiment with P450EryF showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450cin 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 P450EryF, 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 P450cin is controlled.