Cytochrome P450 catalyzed nitric oxide synthesis: a theoretical study.

Similar to nitric oxide synthase (NOS) cytochrome P450 isoforms (e.g. 3A and 4E) can produce nitric oxide from arginine. Although the active site of both proteins contains a protoporphyrin IX unit having an axial cystein ligand, their effectiveness in the synthesis of NO differs significantly. Now the molecular basis of this functional difference was investigated. A homology model for cytochrome P450 3A4 was refined and compared to the X-ray structure of iNOS. We found the active site of iNOS to be more readily accessible for the substrate than that of P450. Docking calculations were performed using the Monte Carlo conformational analysis technique on all internal and external degrees of freedom of arginine and active site residues as well. The lowest energy conformation of the cytochrome P450 3A4-substrate complex was compared to the high resolution X-ray structure of the iNOS-arginine complex. Comparison of substrate orientations revealed that arginine binds in a similar conformation in both enzymes. In contrast to iNOS we found, however, that in P450 partially negative propionate side chains of protoporphyrin IX are located on the opposite side of the heme plane. As a result of this and the absence of other negatively charged residues the distal (substrate binding) side of P450 should be less negative than that of NOS and therefore its affinity toward the partially positive arginine is reduced. Comparison of molecular electrostatic potentials calculated within the active site of the proteins supports this proposal. Reduced affinity in combination with limited substrate access might be responsible for the less effective NO synthesis of cytochrome P450 observed experimentally.