Formation of High-valence Metal-species during Reactions

Chelated transition-metals are known to form high-valent intermediates with peroxides. Also PN behaves alike and it has been previously shown that PN forms ferryl intermediates (Compound II, Fe^IV=O) during reactions with iron (III) porphyrins [103] and Mn^IV/V=O with manganese (II) and (III) porphyrins [174, 50]. Also peroxidases such as HRP and myeloperoxidase (MPO) have been reported to perform similar reactions [102].

Our first observations with Fe(III)edta and PN. Fig.59 showed similar intermediate bands in the reaction of Fe(III)edta with either hydrogen peroxide (left) or PN (right). These bands disappeared faster if an antioxidant such as ascorbate was added. Hence Fe(III)edta also increased the yield of phenolic

nitration by PN, this intermediate must represent the reactive species, during Fe(III)edta-catalyzed nitration.

Figure 59: Reaction of 1 mM Fe(III)edta with either 2 mM H₂O₂(left side) or 800µM PN (right side) at pH 7. Both spectra show the absorbance at around 500 nm before addition of peroxide and 0, 5 and 10 min after the addition.

HRP was the only heme-enzyme which could form a more or less sta-ble ferryl-intermediate during reaction with PN. Bolus-added as well as simultaneously-generated PN caused a shift of the Soret band of HRP at 398 nm to a new intermediate band at 424 nm, which was the ferryl (Compound II) intermediate as previously described [102]. Fig.60A shows the time-dependent accumulation of the ferryl-species during decompostion of SIN-1, which is known to form simultaneously ·NO and O^·−₂ by autoxidation and serves as a source of continuously-generated PN [22]. This ferryl-species could be converted back to the ferric-heme by treatment with ascorbate, indicating the reactivity of this intermediate towards e-donors. This observation was in agreement with the increased nitration of phenol and the accelerated decomposition of PN in presence of HRP. Fig.60B shows the Fe-NO-complex of HRP, which is hard to distinguish from the ferryl-species, but shows no reactivity towards ascorbate.

Bolus addition of either PN or H₂O₂ resulted in similar spectra, suggesting that also in these cases the ferryl-species was formed (not shown). Fig.61 shows the visible region of the ferryl species with the characteristic double bands at 525 and 557 nm. Once more the Fe-NO-complex of HRP showed similar absorbance maxima (not shown). P450’s, hemin and MP-11 formed no stable ferryl-species with PN which could be observed by conventional spectroscopy.

Stopped-flow spectra of hemin and MP-11 revealed only small spectral changes and a decomposition of the porphyrin-ring.

After the finding that P450’s could catalyze the nitration of phenol by PN, we

4 RESULTS 80

Figure 60: Difference absorbance spectra, measured against 5µM native HRP: (A) incu-bated with 400µM SIN-1 at pH 7.4 for 1-13 min (black lines) and 0.5 or 2 min after addtion of 200 µM ascorbate (broken grey lines). (B) incubated with 25 µM DENO at pH 7.4 for 0.2, 1 and 2 min (black lines) and 0.5 or 2 min after addition of 200µM ascorbate (broken grey lines).

Figure 61: Visible region of the HRP ferryl-species generated during the reac-tion of 5µM HRP with 650µM PN at pH 7.5.

concentrated on the mechanism of metal-catalyzed nitration of phenol. Since we found dimerization products also in metal-containing reactions, the mechanism was likely to proceed via radical species and to involve a ferryl intermediate, which we postulated for NOR and PN [163]. As shown in Fig.62 we observed an intermediate during reaction of NOR with PN which could be assigned to the ferryl-species in agreement with the Compound II spectrum of CPO [239].

This ferryl-band increased until most of the PN had decayed (first 100 ms in the kinetic trace in Fig.62) and then started to decompose and formed back the ferric form of NOR (more than 2000 ms). The bimolecular rate constant k_sec for the formation of the ferryl-species was 8x10⁴-2x10⁵ M⁻¹s⁻¹, k_obs was 20 s⁻¹ at pH 7.2 and 43 s⁻¹ at pH 5.9 (see Fig.64). The monomolecular rate constant k_obs for the decomposition was 1.4-1.6 s⁻¹. In the pH range shown the rate constant decreases, but at pH 8.8 it increases again (not shown). From the short life-time we could conclude that Compound II of P450_NOR (and also other P450’s as will be shown later) is much more reactive compared to that one of HRP, which is stable for at least minutes. This also explains the high efficiency of P450’s in catalyzing PN-decomposition and phenol nitration by PN.

Figure 62: Stopped-flow spectra of the reaction of 1.25µM NOR and 250µM PN at pH 7.1. The inset shows the kinetic trace for the decrease and reformation of the Soret band at 417 nm, the formation and decay of a ferryl species at 434 nm and the isosbestic point at 420 nm.

Figure 63: Visible region of the stopped-flow spectra of the reaction of 1.25 µM NOR and 250 µM PN at pH 7.1.

Figure 64: Rate constants for the reac-tion of 2.5 µM NOR with 275 µM PN at different pH.

4.4 Autocatalytic Tyrosine Nitration of Cytochrome P450

and its F87Y Mutant by Peroxynitrite

4.4.1 Short Introduction

In order to explain the high selectivity of the enzyme for nitration, we have used chemical models [130] which suggest that a tyrosine at the active site is nitrated after a homolytic cleavage of PN and a ferryl species as well as nitrogen diox-ide are generated [163]. Interestingly, we have also shown that the P450 protein NADH-NO-reductase (P450_NOR) can catalyze the nitration of added phenol but does not show autocatalytic protein nitration at low PN concentrations [163]. In contrast, the monooxygenase P450BM−3[240] gave a positive reaction with NT-Ab in Western blots after treatment with low PN concentrations [116]. This finding prompted the present study on the location of nitrated tyrosines in P450_BM−3 and in a mutant in which a phenylalanine located close to the active site was exchanged against a tyrosine (F87Y) [182]. This protein displays similarities to

4 RESULTS 82 PGI₂-synthase and can thus be used to study mechanistic aspects on PN medi-ated nitrations, in particular the role of the heme in this process. In addition, since Pfeiffer and Mayer [22] had reported the lack of tyrosine nitration by si-multaneously generated ·NO and O^·−₂ under physiological conditions, we in this work emphasize that this is not valid when tyrosines are in a protein close to a metal-containing active site. In this case reaction with the metal centers can compete effectively with other chemical processes of PN such as isomerization to nitrate or dismutation to dioxygen and dinitrogen trioxide. Uncatalyzed nitration reactions are rather inefficient and may require 100µM or more PN and are thus less likely to occur in cellular systems.