Caught in the act

The isolation of pure quantities of the active intermediate in cytochrome P450 enzymes — known as compound I — has led to this elusive species being characterized in detail.

Credit: © 2010 AAAS

The cytochrome P450 family of enzymes are widespread in nature and involved in the bioactivation and metabolism of the majority of pharmaceuticals. They are of further interest to chemists because of their ability to hydroxylate C–H bonds, which remain stable and unreactive in lab-based organic chemistry. Although the highly reactive intermediate suggested to be responsible for these reactions, known as compound I, is thought to be an iron(IV)oxo species, it has so far proved elusive to full detection and characterization.

Now, Jonathan Rittle and Michael Green from Pennsylvania State University have been able to perform¹ a spectroscopic and kinetic study on compound I as it hydroxylates a C–H bond in lauric acid. They were able to generate compound I in high yields from a very pure sample of the cytochrome P450 enzyme CYP119 from a thermophilic species Sulfolobus acidocaldarius. Rittle and Green were then able to study the elusive intermediate in action using a range of spectroscopic techniques, including Mössbauer, electron paramagnetic resonance and optical absorption. Mössbauer studies confirmed that compound I contains an iron(IV)oxo centre exchange-coupled to a radical porphyrin ligand.

The kinetic characterization revealed that the rate constant for the oxidation of lauric acid was 1.1 × 10⁷ M⁻¹ s⁻¹, which Rittle and Green call 'remarkably efficient'. A large kinetic isotope effect was measured, supportive of values and a suggested mechanism from previous studies.