A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon–hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C–H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron–electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The str...