Nitration and inactivation of cytochrome P450(BM-3) by peroxynitrite - Stopped-flow measurements prove ferryl intermediates

Peroxynitrite (PN) is likely to be generated in vivo from nitric oxide and superoxide. We have previously shown that prostacyclin synthase, a heme-thi olate enzyme essential for regulation of vascular tone, is nitrated and ina ctivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1 996) FEBS Lett. 382, 101-104] and we have studied the effect of heme protei ns on the PN-mediated nitration of phenolic compounds in model systems [Meh l, M., Daiber, A. & Ullrich, V. (1999) Nitric Oxide: Biol. Chem. 2, 259-269 ]. In the present work we show that bolus additions of PN or PN-generating systems, such as SIN-1, can induce the nitration of P450(BM-3) (wild-type a nd F87Y variant), for which we suggest an autocatalytic mechanism. HPLC and MS-analysis revealed that the wild-type protein is selectively nitrated at Y334, which was found at the entrance of a water channel connected to the active site iron center. In the F87Y variant, Y87, which is directly locate d at the active site, was nitrated in addition to Y334. According to Wester n blots stained with a nitrotyrosine antibody, this nitration started at 0. 5 mum of PN and was half-maximal between 100 and 150 mum of PN. Furthermore , PN caused inactivation of the P450(BM-3) monooxygenase as well as the red uctase activity with an IC50 value of 2-3 mum. As two thiol residues/protei n molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitratio n), our data strongly indicate that the inactivation is due to thiol oxidat ion at the reductase domain rather then to nitration of Y residues. Stopped -flow data presented here support our previous hypothesis that ferryl-speci es are involved as intermediates during the reactions of P450 enzymes with PN.