Cooperativity of cytochrome P450 1A2: interactions of 1,4-phenylene diisocyanide and 1-isopropoxy-4-nitrobenzene.

Homotropic cooperativity of 1-alkoxy-4-nitrobenzene substrates and also their heterotropic cooperative binding interactions with the iron ligand 1,4-phenylene diisocyanide (Ph(NC)2) had been demonstrated previously with rabbit cytochrome P450 (P450) 1A2 [G.P. Miller, F.P. Guengerich, Biochemistry 40 (2001) 7262-7272]. Multiphasic kinetics were observed for the binding of Ph(NC)2 to both ferric and ferrous P450 1A2, including relatively slow steps. Ph(NC)2 induced an apparently rapid change in the circular dichroism spectrum, consistent with a structural change, but had no effect on tryptophan fluorescence. Ph(NC)2 binds the P450 iron in both the ferric and ferrous forms; ferric P450 1A2 was reduced rapidly in the absence of added ligands, and the rate was attenuated when Ph(NC)2 was bound. No oxidation products of Ph(NC)2 were detected. Docking studies with a rabbit P450 1A2 homology model based on the published structure of a human P450 1A2.alpha-naphthoflavone (alphaNF) complex indicated adequate room for a complex with either two 1-isopropoxy-4-nitrobenzene molecules or a combination of one 1-isopropoxy-4-nitrobenzene and one Ph(NC)2; in the case of alphaNF no space for an extra ligand was available. The patterns of homotropic cooperativity seen with 1-alkoxy-4-nitrobenzenes (biphasic plots of v vs. S) differ from those seen with polycyclic hydrocarbons (positive cooperativity), suggesting that only with the latter does the ligand interaction produce improved catalysis. Consistent with this view, Ph(NC)2 inhibited the oxidation of 1-isopropoxy-4-nitrobenzene and other substrates.