Freeze frame

Adding oxygen, say in the form of a hydroxyl group, onto a hydrocarbon is not easy. In the absence of catalytic agents, the reaction will proceed only at high temperatures, and even then, the addition of hydroxyl groups may occur at several positions simultaneously. To perform this task at physiological temperature and at specific sites, Nature has designed enzymes, such as those in the cytochrome P450 family, to catalyze a wide range of oxidation reactions of organic molecules, including those involved in steroid synthesis and drug metabolism.

At the active site of a cytochrome P450 is an iron-containing heme group that binds and activates oxygen for the oxidation reaction. Both the molecular oxygen-bound form and the subsequently activated oxygen-bound form of the enzyme are short-lived intermediates in the reaction, making it difficult to directly observe their structures. Now, by collecting X-ray diffraction data at liquid nitrogen temperature on crystals of a cytochrome P450–substrate system (the bacterial P450_cam–camphor complex) trapped at different intermediate stages of the reaction, Schlichting and coworkers (Science, 287, 1615–1622 ) have provided the first snapshots of these intermediate species.

To obtain a stabilized intermediate for structural characterization, Schlichting and coworkers first diffused a reductant, dithionite, into the crystal of the P450_cam–camphor complex to reduce the ferric ion and prime the protein for oxygen binding. They then exposed the crystal to oxygen. Importantly, both steps were performed at low temperature thus trapping the dioxygen bound intermediate.

At the active site of the ternary P450_cam–O₂–camphor complex, one atom in the dioxygen molecule (red) is clearly observed bound to the iron (purple); the other oxygen atom is in close proximity to camphor (green) allowing van der Waal interactions between the two molecules. Two well-ordered water molecules (blue) are in place to form hydrogen bonds with the highly conserved Asp 251 and Thr 252, both of which had been implicated in the catalytic reaction. These two water molecules were absent in the structure of the ferric P450 _cam–camphor complex (no oxygen bound), and could be involved in the oxygen-activation step of the reaction.

The structure of the enzyme intermediate at atomic resolution confirms many conclusions derived from spectroscopic and mutagenesis studies and provides a new reference point for future studies on the reaction mechanism of cytochrome P450. This work also demonstrates the potential of low temperature X-ray crystallography in investigations of enzyme catalysts.