Nitration and inactivation of cytochrome P450BM-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-thiolate enzyme essential for regulation of vascular tone, is nitrated and inactivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1996) FEBS Lett. 382, 101-104] and we have studied the effect of heme proteins on the PN-mediated nitration of phenolic compounds in model systems [Mehl, 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 P450BM-3 (wild-type and 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 located at the active site, was nitrated in addition to Y334. According to Western blots stained with a nitrotyrosine antibody, this nitration started at 0.5 microM of PN and was half-maximal between 100 and 150 microM of PN. Furthermore, PN caused inactivation of the P450BM-3 monooxygenase as well as the reductase activity with an IC50 value of 2-3 microM. As two thiol residues/protein molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitration), our data strongly indicate that the inactivation is due to thiol oxidation at the reductase domain rather then to nitration of Y residues. Stopped-flow data presented here support our previous hypothesis that ferryl-species are involved as intermediates during the reactions of P450 enzymes with PN.
Daiber
A
A
Department of Biology, Universität Konstanz, Germany.
Herold
S
S
Schöneich
C
C
Namgaladze
D
D
Peterson
J A
JA
Ullrich
V
V
D000818:Animals; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase; D009566:Nitrates / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014527:Uric Acid / Q000494:pharmacology
D001426:Bacterial Proteins; D009566:Nitrates; D020030:Nitric Oxide Donors; C039642:peroxynitric acid; D014527:Uric Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D003577:Cytochrome P-450 Enzyme System; D008981:Molsidomine; D007501:Iron; D006899:Mixed Function Oxygenases; D013482:Superoxide Dismutase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D005978:Glutathione; D009584:Nitrogen; D010100:Oxygen; D004229:Dithiothreitol