Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway.

The aim of these experiments was to understand how self-generated HOCl regulates MPO catalytic activity. Our initial experiment utilized H 2 O 2 -selective electrodes to determine whether the accumulation of HOCl in the reaction mixture caused MPO feed-back inhibition. After addition of an aliquot of H 2 O 2 to the continuously stirred reaction mixture (100 mM Cl − final), the H 2 O 2 signal rose rapidly, achieved a maximum after ~30 s, and decreased gradually as H 2 O 2 was depleted by autoreduction (2 H 2 O 2 +O 2 →2 H 2 O) ( Fig. 2 , inset, dashed line). Subsequent addition of MPO (40 nM) to the reaction mixture caused a rapid decay in the level of H 2 O 2 ( Fig. 2 , inset, solid line), indicating that MPO is catalytically active, similar to prior reports [ 37 , 38 ]. We next investigated how multiple rounds of HOCl production would affect MPO catalytic activity and H 2 O 2 consumption, by reversing the order of H 2 O 2 and MPO addition to the reaction mixture. H 2 O 2 -selective electrode measurements revealed that addition of H 2 O 2 (10 μM, final) to a continuously stirred buffer containing MPO (40 nM)/Cl − (100 mM) solution led to an instant disappearance in the H 2 O 2 signal due to its consumption as a substrate by the enzyme during steady-state catalysis ( Fig. 2A ; first trace). Incre-mental additions of the same amount of H 2 O 2 to the reaction mixture inhibited MPO activity, as judged by the accumulation of H 2 O 2 (indicated by the amplitude of the signal) and the decreased rate of H 2 O 2 consumption (indicated by the duration of the signal) ( Fig. 2A ). This reduction in MPO activity required the buildup of sufficient amounts of HOCl that was generated by multiple turn-over of H 2 O 2 consumption. Next, we examined whether HOCl, once generated, first diffuses out from the active site and equilibrates in the solution as a whole, or if the process of inhibition occurs internally without HOCl equilibrating in the solution. Similar experiments were repeated in the presence of a saturated amount of HOCl scavenger (methionine). The rates of MPO-mediated H 2 O 2 consumption in the presence of saturated amounts of methionine were remarkably fast, suggesting that HOCl equilibrates with the solution first before inhibiting MPO ( Fig. 2B ). Alternatively, methionine may destabilize the MPO–Compound I–Cl complex that could be formed during catalysis generating MPO–Fe(III) and chlorinated methionine [ 39 ]. Thus, eliminating HOCl from the MPO milieu by scavenging HOCl and/or inhibiting MPO catalytic activity partially or completely prevent MPO from HOCl insults. To quantitate the extent to which the enzyme was inhibited by self-generated HOCl during steady-state catalysis, we derived estimated standards for H 2 O 2 consumption as a function of decreasing amounts of MPO (10.0–1.25 nM). As depicted in Fig. 2C , addition of 10 μM H 2 O 2 to a continuously stirred buffer solution supplemented with 100 mM Cl − caused a rapid buildup in H 2 O 2 signal and achieved a maximum after ~30 s. Addition of 10 nM MPO to the buffer solution supplemented with 100 mM Cl − , resulting in the production of HOCl, caused a rapid consumption of H 2 O 2 as judged by the disappearance of H 2 O 2 signal. Repeating the same experiment by adding decreased amounts of MPO caused a decrease in the H 2 O 2 consumption rate. By comparing the slope in Fig. 2A with the estimated standards, the degree of MPO inhibition (after five rounds of H 2 O 2 additions) was remarkably high and calculated to be ~10–20% of the total enzyme activity. We next utilized UV–Vis spectrophotometry to determine whether the catalytic inhibition of MPO was due to HOCl-mediated MPO heme destruction. Myeloperoxidase as isolated displays a Soret absorbance peak centered at 430 nm, indicative of the heme content of the enzyme. The effect of incremental additions of H 2 O 2 (20 μM) in the presence of Cl − (100 mM) on the MPO Soret peak was monitored spectrophotometrically ( Fig. 3A ). With each subsequent addition of H 2 O 2 , there was a proportional decrease in the MPO Soret peak, indicating that HOCl-mediated MPO feedback inhibition is associated with MPO heme destruction. To confirm that the MPO heme destruction is due to the buildup of HOCl concentration in the MPO solution mixture, we investigated the effects of exogenous HOCl on MPO heme destruction. Exposure of a fixed amount of MPO (2 μM) to increasing concentrations of HOCl caused MPO heme destruction, as judged by the loss and flattening of the Soret peak region ( Fig. 3B ). Incubation of MPO with 50 μM HOCl caused ~50% decrease in the Soret band, whereas the addition of 200 μM HOCl led to a complete flattening of the Soret absorbance peak, indicating total heme destruction. As MPO heme iron is also known to exist in the ferrous dioxy form (e.g., MPO–Fe(II)–O 2 ; Compound III), we extended these studies to MPO Compound III. As shown in Fig. 3C , addition of molar excess of H 2 O 2 (140 μM) to MPO–Fe(III) in the absence of cosubstrate (Cl − ) caused immediate MPO Compound III formation, as judged by a shift in the Soret absorption peak from 430 to 450 nm and the appearance of additional absorbance peaks in the visible range at 628 nm, as previously reported [ 8 ]. MPO Compound III is relatively stable and required 15 min to decay to the ferric form at 25 °C. Addition of 140 μM HOCl to the MPO solution mixture led to an immediate decrease in the Soret absorbance region, confirming that MPO heme destruction is mediated by HOCl, but not by H 2 O 2 . To confirm that methionine prevents HOCl-mediated MPO heme destruction, a fixed amount of MPO was preincubated with 400 μM methionine followed by subsequent addition of 20 μM increments of H 2 O 2 to the reaction mixture ( Fig. 3D ). Under these circumstances, addition of H 2 O 2 (up to 200 μM in increments of 20 μM) in the presence of Cl − (100 mM) had no significant effect on the MPO absorbance spectra ( Fig. 3D ), suggesting that methionine prevented HOCl-mediated MPO heme destruction. To test the ability of methionine in preventing HOCl-mediated MPO heme degradation, 1.5–2 μM MPO was preincubated with increasing concentrations of methionine (0–100 μM) in 1 ml (final volume) phosphate buffer solution, pH 7.4, supplemented with 100 mM Cl − , and then the reaction mixtures received successive aliquots of H 2 O 2 (20 μM increments). After each addition, the full absorbance spectrum (from 300 to 700 nm) of the reaction mixture was collected, and the percentage recovery in the MPO Soret absorbance peak was plotted as a function of H 2 O 2 concentration. Fig. 3D , inset, shows the plot of percentage destruction (as judged by the loss in absorbance at 430 nm) as a function of H 2 O 2 for various methionine concentrations. As shown in the inset, at all concentrations tested methionine offered some protection early on, but as HOCl started to accumulate (at higher concentrations of H 2 O 2 ), it overcame the protective effect of methionine, and heme destruction was observed. Thus, it can be concluded that methionine prevented self-generated HOCl-mediated MPO inactivation by preventing MPO heme destruction. We next determined if the presence of a 1 e − substrate (e.g., SCN − and NO 2 − ) can compete with HOCl and prevent HOCl-mediated MPO damage. To address this question, we performed experiments utilizing UV–visible spectroscopy to determine whether the MPO/H 2 O 2 system can use SCN − as a 1 e − substrate and prevent MPO heme destruction mediated by self-generated HOCl during catalysis. The experiment was carried out by incremental additions of H 2 O 2 (10 μM) to a MPO–Fe(III) (1.5 μM) solution supplemented with 100 mM Cl − and 100 μM SCN − . Remarkably, subsequent additions of 10 μM H 2 O 2 to the enzyme solution mixture caused an immediate decrease in the Soret absorbance region at 430 nm, with an appearance of a new Soret absorbance peak at 450 nm, indicating that SCN − is utilized as a 1 e − substrate by MPO during catalysis. The generated Compound II was unstable and gradually returned to MPO–Fe(III) after all H 2 O 2 was consumed by the enzyme. Similar results were obtained when SCN − was replaced with NO 2 − (100 μM), whereas replacement with Br − (100 μM) did not show any sign of protection against HOCl destruction. These results indicate that SCN − /NO 2 − , but not Br − , inhibits HOCl production. Change in Cl − concentration within these ranges (80–500 mM) has little or no effect on MPO feedback inhibition. Higher concentration of Cl − serves as a ligand for MPO–Fe(III) leading to enzyme inhibition through the formation of a low-spin six-coordination MPO–Fe(III)–Cl complex. Higher levels of Cl − (1–5 M) caused a shift to the right in the curve of the percentage of MPO heme destruction versus H 2 O 2 concentration, indicating that Cl − is bound to the MPO heme iron moiety, and less HOCl is generated in the enzyme milieu. We next utilized diode array stopped-flow spectrophotometry to continuously monitor and identify species that were formed upon mixing the MPO solution with phosphate buffer supplemented with increasing concentrations of exogenous HOCl. All HOCl concentrations employed were in larger molar excess to MPO to ensure pseudo-first-order conditions. Fig. 4 shows spectra collected over time for the reaction of MPO with HOCl (12.5–200 μM, final). As shown in Fig. 4 , the starting spectrum demonstrated a characteristic Soret peak at 430 nm with absorbance shoulders at 500 and 631 nm, indicative of ferric heme. Mixing the enzyme solution with 12.5, 50, 100, or 200 μM HOCl caused an immediate decrease in the Soret absorbance region, which was attributed to the formation of MPO–Fe(III)–OCl intermediate. The MPO–Fe(III)–OCl complex displayed a distinct Soret absorbance peak at 434 nm, which could be easily differentiated from the Soret absorbance peaks of MPO–Fe(III) (430 nm) and MPO Compounds I (428 nm) and II (450 nm). The stability and accumulation of this complex depends mainly on the concentration of HOCl. At low HOCl concentrations (12.5–50 μM) MPO–Fe(III)–OCl is converted to MPO Compound II via the formation of Compound I, by an intramolecular electron rearrangement as reported earlier [ 20 ]. Compound I/II is unstable and the majority of the complex that was formed slowly decays back to MPO–Fe(III), over a period of ~5 s after initiating the reaction. At higher HOCl concentrations (>50 μM), however, the spectral transformations were distinctly different. Diode array analysis revealed the accumulation of MPO–Fe(III)–OCl complex, its conversion to Compound II, and the subsequent heme destruction. Collectively our results showed that the pivotal first step in the reaction between MPO–Fe(III) and HOCl is the formation of a MPO–Fe(III)–OCl complex, which is then converted to Compound II. Compound II can subsequently decay either to MPO–Fe(III) (at low HOCl concentration) or to the irreversible heme degradation pathway (at higher HOCl concentrations). Single-wavelength stopped-flow measurements were performed to determine the critical concentration of HOCl beyond which HOCl switches its role from mediating destabilization of Compound I/II to MPO heme destruction. The increase or decrease in absorbance followed at 430 and 450 nm was fitted to a one-exponential function, and the observed rate constant of Compound I/II decay to either MPO–Fe(III) or heme destruction was plotted as a function of HOCl concentration. As shown in Fig. 5 , the rate constant of Compound II decay decreased with increasing HOCl concentration, with an inflection point at ~68 μM HOCl to a slower rate. The inflection point shows the ability of MPO to tolerate HOCl accumulation up to a certain level after which MPO heme destruction occurs. Changes in the pH (from 4.0 to 9.0) did not affect the HOCl feedback mechanism, as judged by UV–visible and H 2 O 2 -electrode measurements (data not shown). This observation may indicate that the amount of HOCl generated by the remaining active enzyme in this pH range is sufficient to compete with and destroy the MPO heme prosthetic group through a feedback mechanism. To test whether HOCl-mediated MPO feedback inhibition, through heme destruction, is associated with free iron release, MPO (1.2 μM) was preincubated with 100 mM Cl − , followed by the addition of aliquots of H 2 O 2 (in increments of 20 μM) to the reaction mixture. The free iron released was measured using ferrozine as detailed under Materials and methods, before and after the addition of 200 μM H 2 O 2 . As shown in Fig. 6 no free iron was detected before the addition of H 2 O 2, whereas after the addition of H 2 O 2 , in the presence of Cl − , ~0.36 μM free iron was detected.

Our results suggest a complex and interdependent relationship between levels of self-generated HOCl and MPO catalytic activity during steady-state catalysis. Self-generated HOCl regulates MPO catalytic activity by heme degradation and subsequent free iron release, a process that is attenuated by HOCl scavengers. Given the toxicity of free iron, this observation has a wide application in biological systems in which MPO is expressed. Incremental addition of H 2 O 2 to a catalytic amount of MPO resulted in a remarkably high degree of MPO inhibition (~80 to 90%), allowing the enzyme to function at only a fraction of its maximum activity. This decrease in MPO activity was shown to correlate well with the loss and flattening of the MPO Soret peak, as well as the buildup of free iron, suggesting heme destruction as the mechanism of MPO inactivation. In contrast this process did not occur under conditions where MPO began reducing H 2 O 2 without generating HOCl (e.g., in the absence of Cl − , in the presence of a 1 e − substrate that destabilizes compound II, or in the presence of melatonin, a potent inhibitor of MPO [ 6 , 40 – 42 ]), indicating that self-generated HOCl, and not H 2 O 2 , is the cause of MPO inactivation. The fact that HOCl scavengers prevent this process indicates that HOCl first equilibrates in the solution after generation, before heme destruction. Our rapid kinetic measurements clearly showed that Compound II, the long-lived intermediate that is formed through the reaction of HOCl and MPO–Fe(III), is the prime target for HOCl-mediated heme destruction and subsequent free iron release. These findings display an important application in biological systems because enhancement in MPO expression accompanied by increased free iron levels is a characteristic feature of many inflammatory and cardiovascular diseases, as well as various forms of cancers [ 11 , 12 ]. In general, the balance between the presence of a 1 e − substrate (e.g., SCN − , NO 2 − , NO, and melatonin) versus a 2 e − substrate (e.g., H 2 O 2 ) in the MPO milieu is very important for MPO action and prevention of the damage caused by HOCl. These results are consistent with work by Tahboub et al. [ 32 ], and Burner et al. [ 40 ] showed that SCN − and NO 2 − can serve not only as ligands of MPO–Fe(III), but also as 1 e − substrates of MPO Compounds I and II [ 32 , 40 ]. The preincubation of SCN − with MPO affects the enzyme catalytic site, alters heme iron reactivity, and decreases its affinity toward H 2 O 2 . Rapid kinetic measurements indicated that upon mixing MPO preincubated with SCN − , Compound II is the predominant species formed, allowing the enzyme to operate at only a fraction of its maximum activity [ 32 ]. Nitrite is an excellent substrate for MPO Compound I but reacts slowly with Compound II. It is not that “bad” a substrate for MPO Compound II at low pH [ 40 ]. Nitrite oxidation occurs during the peroxidatic cycle of MPO in a two-1 e − oxidation and yields nitrogen dioxide. Nitrite not only acts as electron donor for Compounds I and II, but also forms a low-spin complex with the ferric enzyme [ 40 ]. Collectively, these findings suggest that SCN − /NO 2 − , but not Br − , inhibited HOCl production by competing with Cl − and switching the MPO catalytic activity from a 2 e − oxidation to a 1 e − oxidation pathway. The pH profile of MPO activity shows a bell-shaped pattern, with optimum pH ranging from 5.5 to 7.5, and drops sharply up to ~30% at pH 4.0 and to ~10% at pH 9.0 [ 43 ]. This pH range (4.0–9.0) has little or no effect on the HOCl feedback mechanism, as judged by UV–visible and H 2 O 2 -electrode measurements. This observation may indicate that the amount of HOCl generated by the remaining active enzyme in this pH range is sufficient to compete with and destroy the MPO heme prosthetic group through a feedback mechanism. Therefore, we predict the same to hold true under physiological and pathological conditions (pH 5–8) where HOCl scavengers are lacking. A kinetic model of how HOCl generated by MPO modulates the classical peroxidase cycle is shown in Fig. 1 . In general, the rapid reaction between MPO and H 2 O 2 forming Compound I is considered the first step in the catalytic cycle. Compound I forms HOCl through a one-step two-electron oxidation of Cl − and is reduced back to the ferric state. When HOCl is generated, it first diffuses out of the active site and equilibrates with the solution as a whole, and may influence the steady-state catalysis by two distinct mechanisms: (1) by converting both MPO–Fe(III) and Compound I to Compound II and (2) by acting as a heme ligand to form the inactive MPO–Fe(III)–OCl complex that can only rejoin the productive cycle through the dissociation of OCl − from the complex. Therefore, HOCl may act as a negative feedback modulator of MPO. Although previous studies have focused on the ability of HOCl to interact with MPO–Fe(III), Compound I, and Compound III to form Compound II, neither the role of HOCl as a ligand for MPO nor its role as a potential mediator of MPO heme destruction has been explored. The direct reaction between HOCl versus MPO–Fe(III) or Compound I is fast and occurs with second-order rate constants ranging from 2 × 10 8 [ 44 ] to 2–5 × 10 4 M −1 s −1 [ 45 ]. The present studies clearly showed that self-generated HOCl may serve as a ligand for MPO, leading to catalytic inhibition and formation of a MPO–Fe(III)–OCl complex, which sets the stage for MPO heme destruction and free iron release. At HOCl concentrations less than the dissociation constant for OCl − (<75 μM), accumulation of MPO–Fe(III)–OCl was not observed; instead the accumulation of Compounds I/II and their decay to MPO–Fe(III) was seen. In contrast, at higher HOCl concentrations, a significant increase in the stability and rate of MPO–Fe(III)–OCl formation was observed, which correlated with the proportional increase in the duration of the reaction as determined by the time elapsed during steady-state catalysis. Under these circumstances, OCl − predominantly serves as a ligand for MPO–Fe(III), generating its inactive MPO–Fe(III)–OCl complex ( Fig. 1 ), whereas the rate of MPO–Fe(III)–OCl complex formation significantly exceeds its rate of conversion to Compound I. As the dissociation rate constant of MPO–Fe(III)–OCl is slower than the formation of Compound I, accumulation of Compound I was not seen; instead accumulation of Compound II was observed. When HOCl concentrations were increased, the transition of the pathway of Compound II exhaustion was reflected by the inflection in the decay rate, with a HOCl critical concentration of 68 μM ( Fig. 5 ). Below this concentration, HOCl was capable of destabilizing Compound I/II, but does not lead to heme destruction. Thus, under these circumstances, MPO tolerates HOCl accumulation by acting as a catalytic sink for HOCl. Alternatively, when HOCl accumulates in the reaction milieu, the rate-limiting step becomes the dissociation of OCl − from the MPO–Fe(III)–OCl complex ( Fig. 1 ). Collectively, this indicates that the loss of MPO catalytic activity is due to the buildup of inactive intermediates, Compound II, and MPO–Fe(III)–OCl, and/or MPO heme destruction. Loss of MPO catalytic activity was correlated with MPO heme destruction and was also associated with the buildup of free iron. Experiments that utilized methionine to scavenge HOCl showed that the magnitude of heme destruction directly depends on the HOCl concentration achieved in the reaction mixture. Thus, the presence of HOCl scavengers at sites of infection may play an essential role in maintaining the catalytic activity of MPO and limiting free iron accumulation that is attributed to HOCl-mediated feedback heme destruction. The bioavailability of HOCl and its ability to compete with H 2 O 2 on the active site of MPO–Fe (III) are key features that drive the enzyme to alter its function from peroxidation to a source of free iron. A theoretical detailed chemical mechanism describing the involvement of HOCl in tetrapyrrole macrocyclic compound destruction (e.g., corrin ring, heme, and porphyrin) has been recently shown [ 18 , 20 , 25 ] and could also apply to MPO heme destruction and subsequent free iron release. In these models, we proposed that, after the formation of Compound II, HOCl can oxidatively cleave the heme ring, leading to the generation of pyrrole derivatives of different chain lengths. Upon cessation of HOCl synthesis due to heme destruction, H 2 O 2 buildup and slow autoreduction was observed. Moreover we have shown how hemoproteins such as hemoglobin and lactoperoxidase, in addition to heme degradation, undergo protein aggregation when they react with HOCl [ 19 , 20 ]. Extensive studies by Chapman et al. [ 46 ] showed that HOCl can cause aggregation in apo-hemoglobin by the formation of noncovalent interactions caused by the generation of protein carbonyls. Additionally, prevention of protein carbonyl formation prevented HOCl-induced protein aggregation [ 47 ]. However, the role of self-generated HOCl in MPO protein aggregation is still under investigation. Therefore, events that are associated with elevated levels of the MPO–HOCl system and/or decreased bioavailability of HOCl scavengers such as taurine, methionine, glutathione, and lycopene may highlight a new pathway for the generation of free iron in diverse inflammatory diseases [ 6 , 11 , 12 , 37 , 48 ]. For example, MPO and free iron were not only elevated in ovarian cancer, but they also correlated with the stages of ovarian cancer [ 12 ]. Therefore, the authors suggested that the combination of these biomarkers can be used to provide an accurate method for early detection and prognosis of the disease. Previous studies have shown catalytically active MPO and iron to be present in human atherosclerotic lesions [ 49 – 52 ]. Our current and recently published results provide a potential link between enhanced MPO activity and free iron accumulation [ 18 – 20 ]. Thus, inhibiting MPO and/or eliminating its final products may play a beneficial role by reducing free iron release in a wide variety of inflammatory conditions. MPO can be inhibited by multiple pathways, including destabilization of MPO Compound II (e.g., melatonin and NO) [ 6 , 7 ]; heme reduction that causes collapse or narrowing of heme pocket geometry, preventing the access of the substrate to the catalytic site of the enzyme (e.g., ascorbate) [ 53 ]; switching the MPO catalytic cycle from peroxidation to catalase-like activity (e.g., melatonin, tryptophan, tryptophan analogs) [ 6 , 37 , 54 ]; or direct scavenging of HOCl (e.g., lycopene) [ 48 ]. MPO plays an essential role in bacterial killing by generating lethal amounts of HOCl within the neutrophil phagosome that forms under pathophysiological conditions [ 55 ]. Although the total amount of MPO in unstimulated neutrophils is shown to be 3.3±0.3 μg (44±4 pmol)/10 6 cells, the amount of MPO generated from phagosomes is mainly dependent on the number of granules and has been reported to reach a concentration of 1–2 mM [ 55 ]. Therefore, at the site of infection, generation of large amounts of cytotoxic HOCl is expected. High concentrations of HOCl could affect bacterial growth by an iron sequestration mechanism through its ability to mediate hemoprotein heme destruction and subsequent iron release that leads the invaded host to withhold iron from bacteria. The generation of free iron under pathological conditions may protect the host from frequent bacterial infections by • OH generation through an H 2 O 2 -driven Fenton reaction [ 55 – 57 ]. The effects of iron release may be much more important inside the phagolysosome than outside it, where the iron-binding capacity is much higher (because of the large pool) and the levels of both MPO and H 2 O 2 are lower. Extracellular ferric iron is insoluble under aerobic conditions and neutral pH and hence cannot be accessed by invading pathogens, greatly decreasing the virulence of some pathogens [ 55 ]. In addition, a substantial proportion of neutrophil MPO (12 pmol with 210 min of stimulation) is released during neutrophil extracellular trap (NET) formation, which corresponds to 30% of the total cellular content, with a further 5% free in the medium. Parker and Winterbourn [ 58 ] have measured the amount of MPO retained by the stimulated neutrophils and found that it accounted for most of the difference between total and released MPO, implying that little of the MPO was inactivated during NET release. Several other groups have attempted to estimate the amount of MPO present on extracellular bacteria. Britigan et al. [ 59 ] have shown that incubation of MPO with certain strains of bacteria (e.g., Escherichia coli and Pseudomonas aeruginosa ) resulted in stable association of MPO with the bacteria, which enhanced their susceptibility to killing by H 2 O 2 . MPO activity ranged from 60 to 204 o -dianisidine U/10 10 organisms (median=100 U/10 10 organisms, n =7) for the E. coli and 6.7 to 53 o -dianisidine U/10 10 organisms for the P. aeruginosa strains (median=13.2, n =7). It is also important to remember that released heme would be free for a very short time, as hemopexin (a plasma protein) rapidly binds heme and delivers it to cells via a hemopexin receptor [ 59 ]. Neutrophil cytoplasm contains around 80 mM Cl − , with a variety of Cl − channels that provide access to the phagosome [ 60 ]. Soluble agonists, as well as particle ingestion, can cause rapid Cl − efflux into the surroundings through specific channels [ 55 , 60 ]. The outward pumping of cytoplasmic Cl − by stimulated neutrophils may be essential for preserving enough phagosomal Cl − levels for HOCl production [ 55 , 61 ]. Chloride is also necessary for azurophil degranulation, and this may be a means of limiting MPO release when Cl − is exhausted [ 55 , 62 ]. The Cl − concentration used in our study was chosen to equal that of the plasma levels (100 mM). Change in Cl − concentrations within these ranges (80–500 mM) has no effect on MPO feedback inhibition. High concentration of Cl − may also serve as a ligand for MPO–Fe(III) leading to the enzyme inhibition through the formation of a low-spin six-coordination MPO–Fe(III)–Cl complex. Thus, the rate-limiting step in MPO catalysis becomes the relatively slow dissociation of Cl − from the complex [ 54 ]. Examination of the H 2 O 2 concentration dependence for MPO heme destruction mediated by self-generated HOCl revealed that the curve shifted to the right. A shift to the right indicates that Cl − is bound to the MPO heme iron moiety, and less HOCl is generated in the enzyme milieu. In summary, MPO binding of self-generated HOCl partially destroys its catalytic site, which in this case is the heme moiety. This alone may be important in biologic settings in which MPO is expressed. The fact that HOCl-mediated heme destruction is largely dependent on the H 2 O 2 in solution adds another level of complexity because the cellular environment can control the extent of HOCl regulation of MPO. Thus, any changes that alter cellular HOCl scavengers or exposure to HOCl scavengers such as melatonin, taurine, glutathione, or methionine will potentially affect HOCl levels and, in turn, alter HOCl-mediated MPO heme destruction and subsequent free iron release. Hypochlorous acid-mediated changes in MPO function are irreversible and dynamic and occur in concert with changes in environmental H 2 O 2 and HOCl concentrations.

All the materials used were of the highest grade purity and used without further purification. Sodium hypochlorite (NaOCl), ammonium acetate (CH 3 COONH 3 ), ferrozine, melatonin, L-methionine, taurine, and ascorbic acid were obtained from Sigma–Aldrich (St. Louis, MO, USA). Myeloperoxidase was initially purified from detergent extracts of human leukocytes by sequential lectin affinity and gel-filtration chromatography [ 30 – 32 ]. Trace levels of contaminating eosinophil peroxidase were then removed by passage over a sulfopropyl Sephadex column [ 31 ]. Purity of isolated MPO was established by demonstrating a Reinheitzal value of >0.85 ( A 430 / A 280 ), SDS–PAGE analysis with Coomassie blue staining, and gel tetra-methylbenzidine peroxidase staining to confirm no contaminating eosinophil peroxidase activity. Enzyme concentration was determined spectrophotometrically utilizing extinction coefficients of 89,000 M −1 cm −1 /heme of MPO [ 33 ]. The concentration of the MPO dimer was calculated as half the indicated concentration of heme-like chromophore [ 34 ]. Hydrogen peroxide consumption was measured using an H 2 O 2 -selective electrode (Apollo 4000 free radical analyzer; World Precision Instruments, Sarasota, FL, USA). Experiments were performed at 25 °C by immersing the electrode in 3 ml of 0.2 M sodium phosphate buffer, pH 7.4. H 2 O 2 (10–20 μM) was added to a continuously stirred buffer solution containing fixed (40 nM, final) or various concentrations of MPO (1.25–40 nM, final) and/or Cl − (100 mM) during which the change of H 2 O 2 concentration was continuously monitored. The absorbance spectra were recorded using a Cary 100 Bio UV–visible spectrophotometer, at 25 °C, pH 7.0. Experiments were performed in 1 ml phosphate buffer solution supplemented with fixed amounts of MPO (1.0 μM) and Cl − (100 mM) and increasing concentrations of H 2 O 2 (0–200 μM), in the absence and presence of sodium thiocyanate (NaSCN), sodium nitrite (NaNO 2 ), or sodium bromide (NaBr) (100 μM). To study the effect of HOCl on MPO heme destruction, similar experiments were repeated in 1 ml phosphate buffer solution supplemented with a fixed amount of MPO (1.0 μM) and increasing concentrations of HOCl (0–200 μM). After 10-min incubation for reaction completion, methionine (fivefold the final HOCl concentration) was added to eliminate excess HOCl and absorbance changes were recorded from 300 to 700 nm. The kinetic measurements of HOCl-mediated MPO heme destruction were performed using a dual-syringe stopped-flow instrument (Hi-Tech Ltd., Model SF-61). Measurements were carried out under an aerobic atmosphere at 10 °C after rapid mixing of equal volumes of a buffer solution containing a fixed amount of MPO (2.0 μM) and a buffer solution containing increasing concentrations of HOCl (0–200 μM). The time course of the absorbance change was fitted to a single-exponential ( y= 1 − e − kt ) or a double-exponential ( y = Ae − k 1 t + Be − k 2 t ) function as indicated. Signal-to-noise ratios for all kinetic analyses were improved by averaging at least six to eight individual traces. In some experiments, the stopped-flow instrument was attached to a rapid-scanning diode array device (Hi-Tech) designed to collect multiple numbers of complete spectra (200–800 nm) at specific time ranges. The detector was automatically calibrated relative to a holmium oxide filter, as it has spectral peaks at 360.8, 418.5, 446.0, 453.4, 460.4, 536.4, and 637.5 nm, which were used by the software to correctly align pixel positions with wavelength. The effect of pH on HOCl-mediated MPO heme destruction was carried out using 50 mM sodium acetate buffer (pH 5) and 50 mM phosphate buffer (pH 6 to 9). Free iron release was measured colorimetrically using ferrozine, with some modifications [ 35 ]. To 100 μl of the sample (MPO–HOCl reaction mixture) 100 μl of ascorbic acid (100 mM) was added. After 5 min of incubation at room temperature, 50 μl of ammonium acetate (16%) and the same volume of ferrozine (16 mM) were added to the mixture and mixed well. Subsequently, the reaction mixture was incubated for 5 min at room temperature and the absorbance was measured at 562 nm. A standard curve prepared using ammonium Fe(III) sulfate was used for the calculation of free iron concentration. Final concentrations of the additives were as follows: ascorbic acid, 33.33 μM; ammonium acetate, 5.3%; and ferrozine, 5.3 μM. Hypochlorous acid was prepared as previously described, with some modifications [ 36 ]. Briefly, a stock solution of HOCl was prepared by adding 1 ml of NaOCl solution to 40 ml of 154 mM NaCl and the pH was adjusted to around 3 by adding HCl. The concentration of active total chlorine species in solution, expressed as [HOCl] T (where [HOCl] T =[HOCl]+[Cl 2 ]+[Cl 3 − ]+[OCl − ]) in 154 mM NaCl, was determined by converting all the active chlorine species to OCl − by adding a bolus of 40 μl of 5 M NaOH and measuring the concentration of OCl − . The concentration of OCl − was determined spectrophotometrically at 292 nm (ε=362 M −1 cm −1 ). As HOCl is unstable, the stock solution was freshly prepared on a daily basis, stored on ice, and used within 1 h of preparation. For further experimentation, dilutions were made from the stock solution using 200 mM phosphate buffer, pH 7, to give working solutions of lower HOCl concentrations.