Autocatalytic nitration of P450(CAM) by peroxynitrite

Peroxynitrite (PN) gains high selectivity as a physiological oxidizing and nitrating agent through catalysis by metal ions. This was established for t he heme-thiolate (P450) enzyme prostacyclin synthase which was tyrosine nit rated and inhibited at low PN levels [FEBS Lett. 382 (1996) 101]. Other P45 0 proteins reacted in a similar manner and a ferryl species (Compound II) h as been identified as an intermediate during reactions with PN [Nitric Oxid e 3 (1999) 142]. Here we investigated cytochrome P450(CAM) and found that i t catalyzes the decomposition of PN as well as an increased nitration of ph enol. The latter at the expense of phenol hydroxylation is characteristic f or the proton-assisted PN action. PN also caused self-nitration of P450(CAM ) at several tyrosine residues. Two of them, Y-96 and Y-305 were largely pr otected in the presence of the ligand metyrapone. Unlike other heme-thiolat e proteins P450(CAM) did not form distinct spectral intermediates character istic for Compound II. We conclude that P450(CAM) serves as a model for the nitration of prostacyclin synthase with respect to its autocatalytic tyros ine nitration and its prevention by blocking the active site. (C) 2000 Else vier Science S.A. All rights reserved.