Oxidations by Peroxynitrite

The balance between ^•NO and O^•−₂ formation is the crucial variable in the ^•NO/O^•−₂ system. Peroxynitrite will be formed whenever both of the radicals are present, but only when the rates of O^•−₂ formation in an activated cell approaches that of

•NO, the subsequently formed peroxynitrite will lead to thiol oxidation, methionine sulfoxidation and tyrosine nitration. A slight excess of either one of the radicals will result in a distinct reaction pattern.

3.5.3.1 Thiol Oxidation

Although peroxynitrite is a potent oxidant, its actual levels will usually stay in the nanomolar range. Under these conditions, oxidations by peroxynitrite often occur in a metal catalyzed manner, as in the case for tyrosine nitration and oxidation of zinc-containing proteins. Peroxynitrite will react with protein thiols to sulfenic acids which then readily form mixed disulfides with GSH. In the case of zinc fingers, Zn²⁺ will be released after disulfide formation between adjacent Cys residues [51, 157, 158].

Since zinc fingers are abundant in transcription factors and required for DNA binding, their oxidation will prevent transcription. Under these oxidative conditions also DNA strand breaks will prevail, therefore this seems to be a meaningful regulation to prevent reading of the unrepaired DNA. The identification of peroxynitrite as the zinc finger-oxidizing intermediate was achieved in context of this work [51] an will be discussed at the accordant position.

Matrix metalloproteinases (MMP) are a family of zinc-containing endopeptidases, responsible for the degradation of extracellular matrix. They are synthesized as pro-enzymes and are known to be activated by oxidant species including peroxynitrite.

Breakage of the bond between Cys and Zn²⁺ at the catalytic centre is necessary for its activation; this can be achieved by proteolytic cleavage, conformational changes or oxidants like peroxynitrite [159–161]. Both MMP-2 [159] and MMP-8 [160] are

reported to be activated by peroxynitrite. This oxidative activation during conditions of oxidative stress can lead to degradation of the extracellular matrix, linked with tissue injury [159, 161, 162]. But apart from roles of MMPs in these long-term remodeling processes, there is increasing evidence that some MMPs like MMP-2 can also rapidly regulate diverse cellular functions, e. g. platelet activation, vascular tone and attenuation of inflammatory signals. These are regulatory mechanisms fitting in the depicted network of complex redox regulative events, especially considering the conditions were oxidations by peroxynitrite occur—if the systems are shifting slowly from nitrosative conditions to oxidative stress. Besides zinc-thiolate targets, increased levels of⁻OONO can also lead to damage of iron prosthetic groups in enzymes, e. g. the iron-sulphur clusters in aconitase [163–165], accompanied with enzyme inactivation.

3.5.3.2 Methionine Sulfoxidation

Methionine (Met) is one of the most readily oxidized amino acid constituents of proteins [166]. Many oxidants in biological systems will attack Met, besides H₂O₂ and hydroxyl radicals also peroxynitrite. The product of these oxidations is methionine sulfoxide [167], which can be reduced back by methionine sulfoxide reductase. Sul-foxidations are in many cases connected with changes in enzyme activity. Therefore, methionine sulfoxidations are presenting a mechanism of redox regulation. A promi-nent example of this regulatory mechanism presents the redox regulation of proteolysis at the level of antiproteases. Some protease inhibitors are reported to be blocked by Met sulfoxidation during physiological processes [168], giving way for protease activation. This seems feasible during immune response and, more generally, at all kinds of inflammation processes.

3.5.3.3 Nitration

Seminal work byBeckmanet al. [169] andIschiropouloset al.[170] demonstrated the capacity of peroxynitrite to cause protein tyrosine nitrationin vitro and postulated that this nitration could occur likewise in vivo. The formation of 3-nitrotyrosine at protein level in vivo as a result of the combined action of ^•NO and O^•−₂ is now well accepted, whereas the chemical mechanism of protein nitration as well as the relevance in redox regulation caused enduring debates. In context of redox regulation, the specificity of Tyr-nitration as well as a regulatory function of this covalent posttranslational protein modification are essential requirements. Eventually, the reversal of nitration stays an unsolved problem, if seen as a regulatory mechanism.

Regarding the mechanism of protein nitration, two distinct pathways leading to 3-nitrotyrosine (3-NT) have to be differentiated. Peroxynitrite itself is unable to yield Tyr-nitration, despite the fact that 3-NT formation is seen as a biomarker for peroxynitrite or at least ^•NO formation in vivo. Goldstein et al. postulated a free radical pathway of nitration in response to the observation that the Tyr-nitration during simultaneous production of ^•NO and O^•−₂ at physiological conditions causes a reaction profile very distinct from that of a bolus addition of peroxynitrite and the different effects of CO₂ on the 3-NT yield [56]. Based on these observations and on pulse radiolysis studies, a mechanism requiring a tyrosyl radical was developed. The formation of a tyrosyl radical can be the consequence of the oxidative actions of CO^•−₃ or oxo-metal complexes and, to a lesser extent, ^•OH. The Tyr radical itself can react with ^•NO2 to yield 3-NT as well as with^•NO, yielding 3-nitrosotyrosine and followed by a sequential two-electron oxidation to 3-NT.

It is now clear that Tyr-nitration does not occur with any Tyr residue at physiological

−OONO levels but requires catalysis by metal centers [171, 172]. According to this mechanism, peroxynitrite would first form a complex with the transition metal, allowing nitration of a proximal Tyr residue [1, 66, 172]:

Me^red + ⁻OONO −−−−−→ [Me^oxO + ^•NO₂] (41) [Me^oxO + ^•NO₂] + Tyr −−−−−→ Tyr–NO₂ + Me^red (42) So far Mn-SOD [173–175] and prostacyclin (PGI₂) synthase [176] are the only known enzymes which will be nitrated even at very low levels of peroxynitrite, allowing nitra-tion not only at condinitra-tions of severe oxidative stress. This autocatalytic mechanism of Tyr-nitration at the metal center of an enzyme would allow to explain these sensitive nitrations. Zou et al. demonstrated that 50 nM peroxynitrite are sufficient to nitrate PGI2 synthasein vitro [176], and later Malinski et al. reported 150 nM ⁻OONO as sufficient to inhibit PGI₂ formation in intact cells [177]. This nitration was shown to occur solely at Tyr-430 of the bovine enzyme [178] and inhibits enzymatic activity irreversibly. The relevance of a selective nitration and inhibition of PGI₂ synthase was demonstrated byBachschmidet al.in anex vivomodel of endothelial cell activation, leading to vasospasm [179]. Since PGHS is still active under such conditions, PGH₂ will accumulate and bind to the TxA₂/PGH₂-receptor on the surface of VSMC, the antagonistic signal to PGI2, to evoke vessel constriction. Considering that under these conditions ^•NO will be blocked by O^•−₂ , the two main pathways of vasorelaxation are effectively blocked, leading to endothelial cell activation and dysfunction.

A chemical reversibility of Tyr-nitration has not yet been found in the case of PGI2

synthase, and hence, this does not exactly meet the requirements of a mechanism of redox regulation but rather has to be considered as a consequence of oxidative stress. In a biological sense, however, 3-NT formation is a reversible process—PGI₂ synthase exhibits a half-life of approximately 30 h (Graf, Ullrich; unpublished results) and nitrated proteins are subject of increased degradation by proteasomal pathways and will be replaced through protein synthesis. The relevant sources of O^•−₂ required for nitration of PGI2 synthase as well as the pathophysiological consequences of its inhibition are also supporting a view of this nitration as a mechanism of oxidative stress.

Under conditions of severe oxidative stress, not only PGI₂ synthase and Mn-SOD will be nitrated, but additionally, a higher number of enzymes was found in a nitrated state. In an cell culture-model, stimulation by cytokines results in Tyr-nitration of aldolase A, GAPDH and actin [175], whereas the latter was also found nitrated in vivo in a model of sickle cell disease [180]. Likewise, a model of hypoxia/reoxygenation revealed a dynamic pattern of protein nitration in mitochondria [175].