Preparation of Compound I in P450cam: The Prototypical P450

Cytochrome P450(cam) occupies a central position in the heme monooxygenase landscape. For many years, P450(cam) has served as a prototype for understanding structure and function in bacterial, fungal, microsomal, and plant P450s. The enzyme is believed to function through a highly reactive iron(IV)oxo radical species called compound I. While the first glimpses of compound I were observed in P450(cam) over thirty years ago, subsequent attempts to prepare the intermediate for further characterizations have proven unsuccessful. Given that the initial reports provided great promise, the nature of the subsequent failures has been extremely puzzling. Here we detail the preparation of compound I in P450(cam). Using insights gained from previous investigations, we have obtained the intermediate in approximate to 45% yield, allowing characterizations by UV/Visible, Mossbauer, and EPR spectroscopies. This report provides a missing piece of the monooxygenase landscape, verifying the existence of compound I in the most studied P450.