Elucidation for the catalytic function of cytochrome P450cam by site-specific incorporation of natural and unnatural amino acid.

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Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the reaction : d-camphor + NADH + H^+ + O_2 * 5-exo-hydroxycamphor + H_2O + NAD^+. In this reaction, 2 electrons from NADH are deliveredto P450cam via putidaredoxinreductaseand putidaredoxin (Pdx). P450cam is reduced by Pdx at the step where it is in the ferric and subsequent oxy-ferrous states.During two years of this project, we have found that a surface residue, Arg112 in P450cam is crucial for the first and second reduction steps. Kinetic studies suggests that : 1) Arg112 forms the binding site for Pdx. 2) Arg112 is an important residue for intramolecular electron transfer (Pdx-P450cam) and also for controlling redox potentials of the P450cam heme-moiety. Another important results of this project is that we have successfully incorporated site-specifically unnatural amino acid, 0-methyl-threonine to the 112 position of P450cam. Catalytic activity measurement of this mutant shows that the mutant enzyme incorporates all the oxygen atom of molecular dioxygen consumed to 5-exo-position of d-camphor, although oxygen consuming activity is one third of that of the wild-type enzyme. This results indicate that the hydroxygroup of threonine is not indispensable for the monooxygenation reaction.We previously showed that monooxygnation of d-camphor was only accomplished efficiently when the hydroxygroup is at 112 position of P450cam. Based on these and other results, we proposed a proton donor and/or acid catalysis as a role of Thr252. In order to validate this proposed role of Thr, we have planned this project and showed successfully the importance of the site-specific incorporation of unnatural amino acid to elucidate the catalytic mechanism of the enzyme.