1 Determinations were carried out at room temperature
1 Determinations were carried out at room temperature
Competition approaches: The kinetics of the reaction between peroxynitrite and Prxs have also been resolved by analyzing the effect of increasing concentrations of the enzyme on another rapid and direct peroxynitrite-dependent oxidation, that of Mn111 porphyrins to O = MnIV porphyrins (Ferrer-Sueta et al, 1999).
(11) ONOO- + Mn111 Porphyrin ^ O = MnIV Porphyrin + NO2
This reporter reaction has the advantages that MnIII porphyrin oxidations can conveniently be followed at the Soret band and the most adequate of a variety of Mn111 porphyrins with different reactivities towards peroxynitrite can be selected for the experiments. In case of MtTPx, MnIII-meso-tetrakis[(N-butyl)pyridinium-2-yl] porphyrin (MnmTB-2-PyP) that reacts with peroxynitrite with a k of 4.8 x 106 M-1s-1 at pH 7.4 and 25 °C, was selected, and the effect of increasing concentrations of reduced MtTPx on the yields of peroxynitrite-mediated MnIIITB-2-PyP oxidation were studied by stopped-flow techniques (Ferrer-Sueta et al., 1999). As shown in Fig. 7, the percentage of inhibition of peroxynitrite-dependent MnIIITB-2-PyP oxidation by MtTPx fitted to a hyperbolic function from which a concentration of reduced MtTPx that half-inhibited MnIII-TB-2-PyP oxidation was determined. At that enzyme concentration, one half of the peroxynitrite reacts with MnIIITB-2-PyP and the other half with MtTPx. Hence, the second order rate constant for the reaction between peroxynitrite and MtTPx, k(MtTPx), can be calculated according to eq. 12:
where k(Mnm-TB-2-PyP) is the second order rate constant for the reaction between peroxynitrite and the MnIII porphyrin under the same experimental conditions. By
Figure 7. Kinetic analysis of the reaction between peroxynitrite and MtTPx by a competition pre-steady state approach. A) MnmTBPyP (7 |M) was exposed to peroxynitrite (2 |M) in potassium phosphate buffer 50 mM, pH 7.35 and 25 ° C, in the presence of the indicated concentrations of Mycobacterium tuberculosis thioredoxin peroxidase (MtTPx), and changes in absorbance, corresponding to Mn111 porphyrin oxidation, were followed at the Soret band by stopped flow technique. B) Percentage of MnmTBPyP oxidation was calculated as 100-(yields of MnmTBPyP oxidation in the presence of MtTPx/yield of MnInTBPyP oxidation in the absence of MtTPx) x 100. Fitting the data to a hyperbolic function, the concentration of MtTPx (3.8 |M) that half inhibited peroxynitrite-dependent MnIIITBPyP oxidation was obtained time (s) [MtTPx] (^M)
Figure 7. Kinetic analysis of the reaction between peroxynitrite and MtTPx by a competition pre-steady state approach. A) MnmTBPyP (7 |M) was exposed to peroxynitrite (2 |M) in potassium phosphate buffer 50 mM, pH 7.35 and 25 ° C, in the presence of the indicated concentrations of Mycobacterium tuberculosis thioredoxin peroxidase (MtTPx), and changes in absorbance, corresponding to Mn111 porphyrin oxidation, were followed at the Soret band by stopped flow technique. B) Percentage of MnmTBPyP oxidation was calculated as 100-(yields of MnmTBPyP oxidation in the presence of MtTPx/yield of MnInTBPyP oxidation in the absence of MtTPx) x 100. Fitting the data to a hyperbolic function, the concentration of MtTPx (3.8 |M) that half inhibited peroxynitrite-dependent MnIIITBPyP oxidation was obtained means of an independently determined k(MnmTB_2_PyP) of 4.8 x 106 M-1s-1 at pH 7.4 and 25 °C [consistent with previous investigations performed at 37 °C (Ferrer-Sueta et al., 2002)] a k(M(TPx) of 1.5 ± 0.2 x 107 M-1s-1 was thus obtained (Jaeger et al., 2004). Similarly, the reactivity between cytosolic TXNPx of T. brucei and peroxynitrite was studied. But since the reaction was slower, a MnIII porphyrin with a lower reactivity towards peroxynitrite, namely, manganese (III)meso-tetrakis [(N-methyl)pyridinium-4-yl] porphyrin (MnmTM-4-PyP) with a k of 3.7 x 106 M-1s-1 at pH 7.4 and 37 °C was selected (Ferrer-Sueta et al., 1999). Increasing concentrations of TbcTXNPx caused a decrease in the yield of peroxynitrite-dependent oxidation of Mnm-TM-4-PyP to O = MnIVTM-PyP and correspondingly an increase of the observed rate constant of peroxynitrite reduction by the TXNPx. From these data a real k of 2 x 106 M-1s-1 at pH 7.4 and 37 °C was calculated, which is similar to the 1 x 106 M-1s-1 value obtained by the direct approach (Trujillo et al. 2004). A similar competitive approach determining the inhibitor effect of Prx on peroxynitrite-mediated horseradish peroxidase oxidation was utilized recently to determine the second order rate constants between two yeast peroxiredoxins and peroxynitrite (Table 2) (Ogusucu et al., 2007). As for other thiol containing compounds, the pH profile for the second order rate constant of peroxynitrite-mediated direct Prx oxidation was bell shaped, led to nitrite formation (Bryk et al., 2000) and to the oxidation of two thiol groups per peroxyni-trite (Trujillo et al., 2004). Moreover, the addition of sub-equimolar peroxynitrite concentrations to reduced bacterial AhpC mutated at the resolving cysteine led to the formation of a sulfenic acid derivative of the peroxidatic cysteine in the enzyme (Bryk et al., 2000). This is consistent with the postulated mechanism of direct peroxynitrite-mediated two-electron oxidations of thiol containing compounds described above.
The availability of direct and indirect methods to determine the kinetics of perox-iredoxin oxidation by peroxynitrite and H2O2, also allows the determination of rate constants for the reaction between reduced peroxiredoxins and other oxidants, such as organic hydroperoxides, whose reduction cannot be directly followed spectropho-tometrically. This method, first employed by Peshenko et al. (2001), determines the inhibitory effect of peroxynitrite or organic hydroperoxides on the peroxiredoxin-dependent hydrogen peroxide consumption; once the second order rate constant for peroxynitrite or hydrogen peroxide and the particular peroxiredoxin is known, the rate constants for other oxidizing substrates of interest can be calculated.
3.3. Steady State Approach: Peroxynitrite Reductase Activities of Peroxiredoxins
In order to reduce peroxynitrite catalytically, Prxs must not only react fast with peroxynitrite, they also have to be oxidized to the same intermediate as by hydroperoxide substrates (Eox1, Fig. 1) to become regenerated by the natural reductant. A qualitative approach to demonstrate catalysis was to study whether the peroxynitrite-oxidized enzyme could be reduced back by disulfide reductants such as DTT which also reduce sulfenic acids (Dubuisson et al., 2004). More compelling evidence for the formation of the presumably physiological sulfenic acid or disulfide intermediate upon peroxynitrite exposure would be the demonstration that the Prx thus oxidized is reduced to the ground state enzyme by its physiological reductant with the rate constant that is observed after oxidation by a hydroperoxide. Therefore, a model system was established that monitored the spontaneous and TXNPx-augmented oxidation of tryparedoxin by peroxynitrite in a stopped-flow equipment. Comparing the experimental traces of peroxynitrite decay with those calculated by computerassisted simulations with predetermined rate constants unequivocally revealed that the peroxynitrite-oxidized TXNPx (of T. brucei and T. cruzi) in this system was catalytically reduced by TXN with rate constants similar to those derived from steady-state analysis (Trujillo et al., 2004). In analogous experiments, the catalytic regeneration of reduced MtTPx from the peroxynitrite-oxidized enzyme was verified (Trujillo et al, 2006).
3.4. Peroxiredoxins Catalytically Detoxify Peroxynitrite Formed from Fluxes of •NO and O2-
All experimental approaches mentioned so far require comparatively high, i. e. unphysiological, concentrations of peroxynitrite. To mimic the effect of Prxs on
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