Conformational and spin-state equilibria in cytochrome P450 BMP: A solid state NMR study

Understanding the structural and dynamic nature of ligand binding to enzymes is of general biochemical interest. Cytochromes P450 constitute a ubiquitous enzyme superfamily that is involved in diverse niches from bioremediation chemistry in bacteria to human biomedicine and drug discovery. The heme domain from cytochrome P450 BM3 (cyt P450 BMP) serves as an ideal model system due to its experimentally amenable nature and sequence similarity to medically relevant isoforms.;Crystallographic studies of cytochrome P450 BMP have indicated that the ligand binds distantly from the heme cofactor. It has been suggested that the binding of ligand to BM3/BMP fits in a sequential process that prepares the BM3/NPG complex for subsequent chemistry, including repositioning of the ligand closer to the heme cofactor after reduction of the heme. We propose instead that the ligand binds to the active site in a temperature-dependent manner, where at low temperature the distal ligand binding mode observed in the crystal structure predominates, while at high temperature alternate proximal ligand binding modes nearer the heme cofactor are primarily occupied.;Optical absorption studies of the BMP/NPG complex show a temperature-dependent spin-state equilibrium from -5° to 35°C, where the high-spin state is dominant at the higher temperatures, then converting to the low-spin state at the lower temperatures. A similar temperature-dependent spin-state equilibrium was also observed in the biomedically relevant human cytochrome P450 3A4 (CYP3A4)/progesterone complex. This is consistent with the proposed "switching" between distal and proximal binding modes as a function of temperature.;Both protein and ligand chemical shifts were obtained for future structural studies of the BMP/NPG complex. One-dimensional 13C solid state NMR studies of the BMP/NPG showed a small linear temperature dependence over approximately a range of 70°C (∼ -35° to 33°C) for the proximally bound ligand. The observed temperature dependence is comparable in sign and magnitude to that observed for the reduced BM3/NPG complex. This implies that the ligand in the proximal state mimics the ligand in the reduced complex, suggesting that reduction is not necessary for ligand rearrangement from the distal to proximal states. Resonance assignments of the Phe509 CO -- Ala510 Calpha pair in BM3, at 48.7 ppm/173.8 ppm, and of Ala82 Calpha, at 51.5 ppm, were obtained by 2D 13C-13C correlation experiments and 1D 15N-13C filter experiments.;High-field NMR studies were carried out with selectively labeled cytochrome P450 BMP complexed with uniform 13C labeled NPG where there is significant spectral overlap from the ligand. We demonstrate that intraligand contacts can be observed, suggesting that future studies with uniform 13C labeled ligand may be feasible for probing the protein-ligand interaction. The viability of rat outer mitochondrial cytochrome b5 as a model paramagnetic metalloprotein for solid state NMR studies was examined, where we were able to detect 13C resonances from the labeled heme cofactor.