Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity

Strong electron-donation from the axial thiolate ligand of cytochrome P450 has been proposed to increase the reactivity of compound I with respect to C–H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we have prepared a selenolate-ligated cytochrome P450 compound I intermediate. This isoelectronic perturbation allows for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepared with meta -chloroperbenzoic acid and characterized by UV–visible, Mössbauer and electron paramagnetic resonance spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we find that the selenolate-ligated compound I cleaves C–H bonds more rapidly than the wild-type intermediate. The oxidative prowess of cytochrome P450s has been suggested to stem from the electron-donating axial ligand. Now, a selenocysteine-ligated P450 compound I has been trapped and characterized providing an avenue to examine this hypothesis. Measurements reveal that the selenolate-ligated compound I cleaves C–H bonds more rapidly than the wild-type equivalent.