Cytochrome P450 catalyzed nitric oxide synthesis: A theoretical study

Similar to nitric oxide synthase (NOS) cytochrome P450 isoforms (e.g. 3A an d 4E) can produce nitric oxide from arginine. Although the active site of b oth proteins contains a protoporphyrin IX unit having an axial cystein liga nd, their effectiveness in the synthesis of NO differs significantly. Now t he molecular basis of this functional difference was investigated. A homolo gy model for cytochrome P450 3A4 was refined and compared to the X-ray stru cture of iNOS. We found the active site of iNOS to be more readily accessib le for the substrate than that of P450. Docking calculations were performed using the Monte Carlo conformational analysis technique on all internal an d external degrees of freedom of arginine and active site residues as well. The lowest energy conformation of the cytochrome P450 3A4-substrate comple x 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, ho wever, that in P450 partially negative propionate side chains of protoporph yrin 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 (subst rate binding) side of P450 should be less negative than that of NOS and the refore its affinity toward the partially positive arginine is reduced. Comp arison of molecular electrostatic potentials calculated within the active s ite 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.