Direct observation of substrate binding to ferrous-CO cytochrome p-450-CAM using 19F NMR

The binding of two fluorinated substrate analogs, 9-fluorocamphor and 5,5-difluorocamphor, to cytochrome P-450-CAM has been investigated by 19F NMR spectroscopy. The NMR properties of each substrate differ depending on whether it is free in aqueous buffer, bound to the diamagnetic ferrous-CO enzyme or bound to the paramagnetic ferrous derivative. As CO must be bound to the ferrous center for it to be diamagnetic, these results demonstrate that camphor and CO bind to be the protein simultaneously. The present results are unusual in that the spectral properties of the substrate rather than those of the heme iron have been monitored to follow substrate and ligand binding. 26 refs.; 2 figs.; 1 tab