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Mechanism of reaction of myeloperoxidase with nitrite
U Burner1, P G Furtmuller, A J Kettle
1Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Abstract:
Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.
Insights
Myeloperoxidase (MPO) utilizes nitrite as a substrate in its enzymatic cycle, particularly during inflammatory conditions. This study elucidates the reaction kinetics and pH dependence of MPO with nitrite.
Area of Science:
- Biochemistry
- Enzymology
- Inflammation Research
Background:
- Myeloperoxidase (MPO) is a key neutrophil protein implicated in inflammatory diseases.
- Tyrosine nitration is observed in inflammatory conditions involving neutrophil and macrophage activation.
- The role of nitrite as a physiological substrate for MPO remains unclear.
Purpose of the Study:
- To investigate the reaction kinetics of MPO with nitrite.
- To determine if nitrite is a physiological substrate for MPO.
- To elucidate the pH dependence of MPO-nitrite interactions.
Main Methods:
- Sequential mixing stopped-flow analysis was employed.
- Reactions of ferric MPO, compound I, and compound II with nitrite were studied.
- Experiments were conducted across a pH range of 4-8 at 15°C.
Main Results:
- MPO forms a low-spin complex with nitrite, with varying rates and dissociation constants at different pH values.
- Nitrite is oxidized by MPO through two one-electron steps.
- Rate constants for the reduction of MPO intermediates (Compound I and II) by nitrite were determined and showed pH dependence.
Conclusions:
- Nitrite is a physiological substrate for MPO, undergoing oxidation within the MPO peroxidase cycle.
- The pH dependence suggests a catalytic residue (likely distal histidine) with a pKa of ~4.3 is crucial for nitrite binding and oxidation.
- These findings contribute to understanding MPO's role in inflammatory processes involving tyrosine nitration.