Models of nitric oxide synthase: Iron(III) porphyrin-catalyzed oxidation of fluorenone oxime to nitric oxide and fluorenone

Nitric oxide synthase (NOS) is a heme-containing monoxygenase that catalyzes the oxidation of L-arginine to L-citrulline and NO in two steps. In the second step of the NOS reaction, citrulline and NO are generated from the heme-catalyzed 3-electron oxidation of L-N-hydroxyarginine. To model this unusual reaction, iron porphyrin-catalyzed oxygenations of oximes with O₂ were investigated. The oxidation of fluorenone oxime and a stoichiometric amount of hydroxoiron(III) porphyrin (Fe(OH)P, P = TMP and TPFPP) with O₂ in benzene generated Fe(NO)P, fluorenone, and O-(9-nitro-9-fluorenyl)fluorenone oxime. The X-ray crystal structure of the oxime ether product suggests that it originated from the dimerization of the fluorenyl iminoxy radicals. Detailed analysis of this reaction showed that the oxime reacted first with Fe(OH)P to generate a 5-coordinate, high-spin oximatoiron(III) porphyrin species [Fe(oximate)P]. The X-ray crystal structure of oximatoiron(III) tetrakis(2,6-dichlorophenyl)porphyrin [Fe(oximate)TDCPP] showed that the oximate ligand was monodentate, O-bound to Fe(III)P. The aerobic oxidation of Fe(oximate)P followed the characteristic kinetics of a metalloporphyrin- catalyzed radical-type autoxidation. O₂ surrogates, the π-acids NO and CO, induced the homolysis of Fe(oximate)P to generate Fe(NO)P or Fe(CO)P and the iminoxy radical, implicating a similar reaction mode for O₂ with Fe(oximate)P. Fe(oximate)TMP reacted with ¹⁸O₂ to generate predominantly ¹⁸O-labeled fluorenone (75% yield), while the reaction conducted under ¹⁶O₂ and H₂¹⁸O generated only ¹⁶O-labeled fluorenone. This reaction is proposed to proceed via an Fe-O bond homolysis of Fe(oximate)TMP followed by O₂ insertion to generate 9-nitroso-9-fluorenylperoxyFe(III)TMP, which decomposes via an O-O bond homolysis to generate NO, fluorenone, and oxoFe(IV)P. The implications of this system for the NOS reaction mechanism are discussed.