Reaction of myeloperoxidase with its product HOCI

Abstract

The reaction of human myeloperoxidase with its product, hypochlorous acid was investigated using both rapid-scan spectrophotometry and the stopped-flow technique. In the reaction of myeloperoxidase with hypochlorous acid a primary compound is found with properties similar to that of compound I and which is converted into compound 11. The primary reaction is strongly pHdependent. At pH 7.2 the reaction is too fast to be measured but at higher pH values it is possible to determine the apparent second-order rate constant. Its value decreases to about 2 x lo7 M-' . s-' at pH 8.3 and to 2.3 (+0.4) x lo6 M -l . s-' at pH 9.2, respectively. The dissociation constant for the formation of the primary compound is 25.7 (f 15.3) pM at pH 9.2 and about 2.5 pM at pH 8.3. The apparent second-order rate constant for the formation of compound I1 is hardly affected by pH and varies between 2 to 5 x lo4 M-' . s-' at pH 10.2 and pH 8.3, respectively.Reaction of myeloperoxidase with hypochlorous acid also resulted in irreversible partial bleaching of the chromophore. Chloride, which is a substrate of the enzyme not only protects myeloperoxidase against bleaching by hypochlorous acid but also competitively inhibits the binding of hypochlorous acid to myeloperoxidase, a process which also has been observed in the reaction with hydrogen peroxide. It is concluded that hypochlorous acid binds at the heme iron to form compound I.Myeloperoxidase (donor :hydrogen peroxide oxidoreductase) is a heme-containing enzyme found in the primary granules of human neutrophils. Because of the unique ability of the enzyme to catalyze the H20z-dependent peroxidation of C1-to HOCI, which is an anti-microbial agent, myeloperoxidase is thought to play an important role in the killing of micro-organisms [I, 21. Several spectroscopically distinguishable forms of myeloperoxidase are known : native enzyme, compounds I, I1 and 111. The first step in the peroxidation of chloride is the reaction of myeloperoxidase with hydrogen peroxide to form compound I [3 -51. Compound I is a short-living intermediate in which the prosthetic group is in an oxidized state which is two oxidation equivalents above the ground state. Compound I is very reactive and able to oxidize chloride to hypochlorous acid. The formation of compound I is a very fast process, the apparent second-order rate constant being 2.3 x lo7 M -' . s- ' [5]. The reaction is pHdependent and its rate is governed by a group which, when protonated, prevents H2O2 from binding to myeloperoxidase. The pK, of this group is 4.3 and it has been suggested that the distal histidine with this pK, governs the binding of H2O2 to the enzyme [5, 61.Chloride is able to inhibit the binding of H z 0 2 to myeloperoxidase competitively [5, 7 -101. This process is governed by the same acid/base equilibrium. However, in contrast to H202, chloride binds to the heme iron when the protonatable group is protonated.Reaction of compound I with reductants, such as D-penicillamine [lf], ferrocytochrome c (121 and also hydrogen peroxide [1...

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

“…It represents the two-electron reduction product of chlorite formed during compound I formation. Generally, HOCl is known to act as two-electron oxidant that transforms ferric peroxidases and catalases to compound I [19]. As expected, in the system HRP/HOCl compound I was formed.…”

“…Hydrogen peroxide concentration was determined spectrometrically using an extinction coefficient at 240 nm of 39.4 M À1 cm À1 [17]. The concentration of sodium chlorite (Sigma, 80%) and hypochlorite were determined using the extinction coefficient at 260 nm of 154 M À1 cm À1 [18] and at 292 nm of 350 M À1 cm À1 [19], respectively. HRP Type VIa was obtained from Sigma and used without further purification.…”

Interaction of myeloperoxidase with peroxynitrite

“…Chloride has been shown to inhibit both the binding of H,O, and that of HOCl to the enzyme [43]. There is considerable evidence that chloride binds to myeloperoxidase at the sixth coordination position of the heme iron [51, 521. This process is governed by a single protonatable group with a pK, of 4-4.7 [42, 53, 541.…”

Chlorite reactivity with myoglobin: Analogy with peroxide and nitrite chemistry?

“…These medical links to globin chemistry provide an additional point of interest for the present investigation, besides generally trying to investigate hemoprotein-hypochlorite interactions with relevance to chlorite dismutases [31], [32] and to some extent haloperoxidases (chloroperoxidase, myeloperoxidase, and others [8], [33], [34].…”