Biochem. J. (2011) 439, 423–431 (Printed in Great Britain) doi:10.1042/BJ20110555 423 Inhibition of the chlorinating activity of myeloperoxidase by tempol: revisiting the kinetics and mechanisms Raphael F. QUEIROZ, Sandra M. VAZ and Ohara AUGUSTO 1 Departamento de Bioqu´ ımica, Instituto de Qu´ ımica, Universidade de S˜ ao Paulo, Caixa Postal 26077, S˜ ao Paulo, CEP 05513-970, Brazil 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 H 2 O 2 , 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 × 10 5 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 [Rees, Bottle, Fairfull-Smith, Malle, Whitelock and Davies (2009) Biochem. J. 421, 79–86]. Key words: chlorinating activity, kinetics, myeloperoxidase, myeloperoxidase inhibition, nitroxides, tempol. INTRODUCTION Nitroxides are stable free radicals that have been extensively used as biophysical tools and are receiving increased attention as potential therapeutic agents because of their pronounced antioxidant properties and low toxicity [1,2]. Under physiological conditions, nitroxides (TPNO • ) almost act as catalytic antioxidants because they react with diverse biological oxidants and reductants during recycling through the oxammonium cation (TPNO + ) and hydroxylamine-derivative (TPNOH) respectively. After several redox cycles, nitroxides are eventually consumed by recombination reactions with specific radicals, such as tyrosyl and thiyl radicals, and/or by metabolism [3–6]. Tempol (4-hydroxy- 2,2,6,6-tetramethyl piperidine-1-oxyl) is a highly investigated nitroxide in vivo and has been shown to effectively reduce tissue injury in animal models of inflammation [7]. The mechanisms of protection are not completely understood, but the antioxidant and anti-inflammatory actions of nitroxides are thought to be related [1,8–10]. An important target for anti-inflammatory compounds is the haemprotein MPO (myeloperoxidase; EC 1.11.1.7) that plays a fundamental role in oxidant production by neutrophils. MPO amplifies the oxidative potential of its co-substrate H 2 O 2 through the oxidizing enzyme intermediates MPO-I [MPO-porphyrin •+ - Fe(IV)=O] and MPO-II [MPO-porphyrin-Fe(IV)=O]. MPO-I contains two more oxidizing equivalents than the resting enzyme [MPO-porphyrin-Fe(III)] and is produced by the rapid reaction between MPO and H 2 O 2 (eqn 1). MPO-II is produced by the reduction of MPO-I (eqn 4) and is a less potent oxidant than MPO-I (MPO-I/MPO-II, E o = 1.35 V; MPO-II/MPO, E o = 0.97 V) (reviewed in [11,12]). MPO + H 2 O 2 ↔ MPO-I + H 2 O + H + (1) MPO-I + Cl - ↔ [MPO-I-Cl − ] H + −→MPO + HClO (2) MPO-I + NO 2 − → MPO-II + NO 2 • + H + (3) MPO-II + NO 2 − → MPO + NO 2 • + H + (4) Among the reactions catalysed by MPO under physiological conditions, the two-electron oxidation of chloride to hypochlorous acid (eqns 1 and 2) has received the most attention because chloride is present at high physiological concentrations and because hypochlorous acid possesses microbicidal activity. The discovery of protein tyrosine nitration in tissues and biological fluids has brought attention to the peroxidase activity of MPO, which oxidizes many biotargets by one-electron mechanisms through the intermediacy of MPO-I and MPO-II (eqns 1, 3 and 4) [11,12]. Nitrite is one of the endogenous substrates of MPO and is oxidized to nitrogen dioxide, particularly at acidic pH [13–15]. The latter oxidizes and nitrates biomolecules, whereas hypochlorous acid oxidizes and chlorinates biomolecules. In addition to being an integral part of the innate immune defense system, emerging evidence has indicated that the oxidants produced by MPO (eqns 1–4) are key mediators of tissue damage associated with many inflammatory diseases such as Abbreviations used: MPO, myeloperoxidase; MPO-I, MPO-porphyrin •+ -Fe(IV)=O; MPO-II, MPO-porphyrin-Fe(IV)=O; tempol, 4-hydroxy-2,2,6,6- tetramethyl piperidine-1-oxyl. 1 To whom correspondence should be addressed (email oaugusto@iq.usp.br). c  The Authors Journal compilation c  2011 Biochemical Society www.biochemj.org Biochemical Journal