Mechanisms of self-catalyzed heme protein nitration by peroxynitrite

Peroxynitrite (PN) is a powerful nitrating and oxidizing agent that has been implicated in a host of cell injuries. Among protein targets in vivo, heme proteins are particularly sensitive to the impairments induced by PN. We present direct evidence that ferrylMb and NO2 are both produced during the interaction between PN and metmyolgobin (metMb). These observations indicate the initial formation of the caged radical intermediate [FeIV=O .NO2] as a precursor of the spectroscopically observed ferrylMb and trappable NO2 in the course of PN-mediated myoglobin nitration. This caged pair [FeIV=O .NO2] mainly rebounds to form metMb and nitrate (k r) in an oxygen rebound scenario. Freely diffusing NO2 is liberated concomitantly from the radical pair (ke) and preferentially nitrates Tyr103 in horse heart myoglobin. The ratio kr/k e was found to be ∼ 10 by examining the nitration yields of fluorescein, an external NO2 trap. We confirm that the nitrating species produced either by metMb/H2O2/NO2 - or peroxynitrite results in the same selective nitration of Tyr 103 in myoglobin. Tyr 103-nitrated myoglobin was purified by anion-exchange HPLC. The overall structure of Mb is preserved upon nitration, whereas the heme configuration is influenced and partially exhibits a transformation from a six-coordinate heme to a five-coordinate heme. Compared to the intact metMb, the peroxidase activity of this nitrated myoglobin is modestly increased by 15%. This observation is consistent with the notion that inhibiting the tyrosine-mediated intramolecular electron transfer pathway may lead to enhanced Mb peroxidase activity. Furthermore, the bimolecular nitroMb/peroxynitrite interaction is decreased one third, possibly due to the steric hindrance and the anionic property of the nitrated Tyr103 located at the entrance of the heme cavity.;Cytochrome c (cyt c) is an electron transfer protein present at high concentrations (∼ 1mM) in the intermembrane space of mitochondria to shuttle electrons between respiratory complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). Cyt c bears a net eight positive charges at neutral pH and binds avidly to acidic phospholipids. These interactions induce conformational changes in cyt c, especially the disruption of the coordination between Met80 and the heme iron. The unique interactions between cyt c and cardiolipin (CL), which can provoke cardiolipin oxygenation under oxidative stress, are required for the release of proapoptotic factors during the initial apoptotic events. We show that cyt c binding to CL induces cyt c conformational transition to a rich beta-sheet structure accompanied with Met80 deligation. Consequently, CL-bound cyt c can react rapidly with peroxynitrite with a bimolecular rate constant 5 x 103 M-1 s-1 when the CL/cyt c ratio reaches 100:1 at pH 7.4 and 25°C. Under this condition, the nitrated tyrosine yield in cyt c increases to ∼45% compared to ∼20% in CL-free cyt c. Kinetic analysis of PN interactions with CL-bound cyt c, associated with fluorescein nitration measurements suggests an .NO2 escape-rebound mechanism similar to that of the metMb and PN reaction. In particular, a distinct NO2 cage escape (∼58%) pathway in cyt c/CL reaction with PN accounts for the appreciable increase in the PN-mediated nitrotyrosine and nitrofluorescein formations. Finally, the results from the tyrosine nitration and methionine oxidation pattern studies support an extended lipid conformation model for the orientation of cyt c on CL-containing membrane surface. In this model, one of the CL acyl chains is accommodated in the hydrophobic cavity of cyt c, while the phosphate head groups of CL interact with Asn or Lys residues electrostatically.