Inhibition of the chlorinating activity of myeloperoxidase by tempol: revisiting the kinetics and mechanisms

Abstract

The nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of inflammation by mechanisms that are not completely understood. MPO (myeloperoxidase), which plays a fundamental role in oxidant production by neutrophils, is an important target for anti-inflammatory action. By amplifying the oxidative potential of H2O2, MPO produces hypochlorous acid and radicals through the oxidizing intermediates MPO-I [MPO-porphyrin•+-Fe(IV)=O] and MPO-II [MPO-porphyrin-Fe(IV)=O]. Previously, we reported that tempol reacts with MPO-I and MPO-II with second-order rate constants similar to those of tyrosine. However, we noticed that tempol inhibits the chlorinating activity of MPO, in contrast with tyrosine. Thus we studied the inhibition of MPO-mediated taurine chlorination by tempol at pH 7.4 and re-determined the kinetic constants of the reactions of tempol with MPO-I (k=3.5×105 M-1·s-1) and MPO-II, the kinetics of which indicated a binding interaction (K=2.0×10-5 M; k=3.6×10-2 s-1). Also, we showed that tempol reacts extremely slowly with hypochlorous acid (k=0.29 and 0.054 M-1·s-1 at pH 5.4 and 7.4 respectively). The results demonstrated that tempol acts mostly as a reversible inhibitor of MPO by trapping it as MPO-II and the MPO-II-tempol complex, which are not within the chlorinating cycle. After turnover, a minor fraction of MPO is irreversibly inactivated, probably due to its reaction with the oxammonium cation resulting from tempol oxidation. Kinetic modelling indicated that taurine reacts with enzyme-bound hypochlorous acid. Our investigation complements a comprehensive study reported while the present study was underway

The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol

“…However, tempol (5-75 μM) had marginal effects on the rate of H 2 O 2 (1 mM) uptake by hSOD1 (30 μM in terms of monomer units) in the presence of bicarbonate (25 mM) ( Figure 1A). In contrast, these concentrations of tempol strongly inhibited the ability of myeloperoxidase to consume H 2 O 2 in the presence of chloride [32,41]. Therefore, the results shown in Figure 1(A) argue against a direct interaction between tempol and the copper ion in the hSOD1 active site.…”

“…The H 2 O 2 solutions were prepared just before use, and the concentrations were determined spectrophotometrically by reaction with horseradish peroxidase to produce compound I ( ε 403 = 5.4×10 4 M − 1 ·cm − 1 ) [31]. The tempol concentration was determined spectrophotometrically (ε 240 = 1.44×10 3 M − 1 ·cm − 1 ) [32]. Mass spectrometry grade trypsin (Gold) was purchased from Promega.…”

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions

“…3). Thus, H 2 O 2 induces protein-centered radical formation on IDO in a manner sensitive to cyanide, which inhibits compound I formation, or tempol, which efficiently inhibits compound I-mediated oxidative reactions by acting as a competitive substrate for this reactive heme species (67,68). Similarly, L-Trp, ascorbate, and tyrosine are all excellent compound I substrates (31,32,53,69), which correlate with their ability to inhibit H 2 O 2 -induced IDO protein oxidation and protect against inactivation of IDO dioxygenase by H 2 O 2 .…”

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

“…Inhibitors that cause accumulation of Compound II are poor peroxidase substrates that react well with Compound I but slowly with Compound II. These include dapsone (5), tryptamines (6), tryptophan analogues (7), and nitroxides (8,9). Such inhibition is unlikely to be effective in a normal physiological environment because of an abundance of better peroxidase substrates such as ascorbate (10) and urate (11) that will efficiently convert any accumulated Compound II back to the active native MPO state.…”