INTRODUCTION:

The Cytochrome P450's comprise a ubiquitous class of heme-containing enzymes involved in the metabolism of xenobiotics. Cytochrome P450cam is used by soil bacterium to hydroxylate the natural substrate camphor. Our interest in P450cam is as a medium for the enzymatic hydroxylation of both natural and synthetic compounds, thus avoiding chemical syntheses involving toxic chemicals. The ultimate goal is to redesign the active site of P450cam through site-directed mutagenesis in order to optimize catalysis of the desired substrates.

Recently, we have examined the binding of (s)-nicotine to P450cam. Nicotine is metabolized by P450cam, resulting in hydroxylation at the 5' position of the pyrrolidine ring. This occurs via attack by an activated oxygen intermediate bound to the heme iron. The structure of the active site of a binary complex between P450cam and (s) -nicotine is shown below:

RESULTS:

The 1-sigma SA-omit 2F0-Fc map at left is from a 2.3 angstrom data set. It shows the nicotine ligand (green) coordinated to the heme iron via its pyridine nitrogen. The resulting model is shown at right. In this orientation, the pyrrolidine ring is "up" away from the heme iron, and could not be hydroxylated by a heme-bound oxygen. This orientation produces a non-productive complex.

In order to mimic the activated oxygen heme ligand, we then introduced CO to the crystals shown above, and recollected data. Results are shown below:

In the presence of a competing heme ligand, the nicotine ring has "flipped" . It is now bound with the pyrrolidine ring "down", close to the catalytic heme group in a more favorable position for hydroxylation:

SUMMARY

1) In the absence of competeing ligands, (S)-nicotine binds to P450cam in a catalyically nonproductive orientation, with the pyridine nitrogen coordinated to the heme iron.

2) Under reducing conditions, and in the presence of an alternative heme ligand like CO, (S)-nicotine binds P450cam in a productive orientation, with the pyrrolidine ring close to the heme group.