INTERACTION OF ANGELI SALT WITH CYTOCHROME-P450 1A2 DISTAL MUTANTS - AN OPTICAL-ABSORPTION SPECTRAL STUDY

Angeli's salt, Na2N2O3 or O--N=N+-(OH)(O-) in aqueous solution, is kno wn to release NO- or NO., which relaxes vascular tissue and lowers blo od pressure. In the liver, the most abundant heme enzyme is cytochrome P450. In the present study, we studied the effect of rat liver cytoch rome P450 1A2 (P450 1A2) in regard to its catalysis of the N=N bond sc ission of Angeli's salt with optical absorption spectra. Also, we exam ined the contribution of putative distal amino acids of P450 1A2 to th e reaction with the salt. We found that wild-type Fe3+ P450 1A2 marked ly enhances the N=N scission of the salt up to 100 fold in terms of ab sorption spectroscopy. A Fe3+ P450 1A2-NO complex with an absorption p eak at 435 nm was formed when the salt was added and the complex was t hen changed to a 6-coordinated Fe2+-NO complex having a 440-nm peak. G lu318Asp, Glu318Ala and Thr319Ala mutants at the putative distal site of P450 1A2 formed a 5-coordinated Fe2+-NO complex having a 400-nm abs orption, that was not formed with the wild type. The Glu318Ala mutant, in particular, did not form the Fe3+-NO complex with the addition of Angeli's salt. The presence of L-Cys, reduced glutathione, catalase or superoxide dismutase markedly stabilized the Fe3+ wild type-NO comple x. Thus, our data suggests that the N=N bond of Angeli's salt is cleav ed with the P450 1A2 active site and NO-or NO. is released. We discuss mechanisms of redox and ligand changes of the P450 heme. (C) 1997 Els evier Science B.V.