Kinetic Evidence Supports the Existence of Two Halide Binding Sites that Have a Distinct Impact on the Heme Iron Microenvironment in Myeloperoxidase

Abstract

Myeloperoxidase (MPO) structural analysis has suggested that halides and pseudohalides bind to the distal binding site and serve as substrates or inhibitors, while others have concluded that there are two separate sites. Here, evidence for two distinct binding sites for halides comes from the bell-shaped effects observed when the second-order rate constant of nitric oxide (NO) binding to MPO was plotted versus Cl- concentration. The chloride level used in the X-ray structure that produced Cl- binding to the amino terminus of the helix halide binding site was insufficient to populate either of the two sites that appear to be responsible for the two phases. Biphasic effects were also observed when the I-, Br-, and SCN- concentrations were plotted against the NO combination rate constants. Interestingly, the trough concentrations obtained from the bell-shaped curves are comparable to normal plasma levels of halides and pseudohalides, suggesting the potential relevance of these molecules in modulating MPO function. The second-order rate constant of NO binding in the presence of plasma levels of I-, Br-, and SCN- is 1-2-fold lower compared to that obtained in the absence of these molecules and remains unaltered through the Cl- plasma level. When Cl- exceeded the plasma level, the NO combination rate becomes indistinguishable from the second phase of the bell-shaped curve that was obtained in the absence of halides. Our results are consistent with two halide binding sites that could be populated by two halides in which both display distinct effects on the MPO heme iron microenvironment.

Potential role of tryptophan and chloride in the inhibition of human myeloperoxidase

“…Since Trp is a bulky molecule and binds on the entrance of MPO heme pocket, there is a direct communication between the active site of the enzyme, where HOCl is generated, and the heme pocket entrance binding (regulatory) site. Our current results are consistent with the hypothesis that Cl − promotes a significant alteration in the heme pocket of ground-state MPO [41,62], as reflected in the observed rates of Compound II formation.…”

“…Early studies by Hori et al have shown that salicylhydroxamic acid and benzohydroxamic acid [64] is a relatively bulky aromatic molecule that may influence MPO steady-state catalysis by binding to the entrance of the hydrophobic pocket of the distal heme cavity, preventing the access of H 2 O 2 to the catalytic site of the enzyme. However, using rapid kinetic measurements and NO binding to MPO heme iron, we have recently shown that Cl − may bind at two different binding sites on MPO [62]. Both sites have distinct effects on the MPO heme iron microenvironment.…”

Melatonin Prevents Myeloperoxidase Heme Destruction and the Generation of Free Iron Mediated by Self-Generated Hypochlorous Acid

“…MPO was initially purified from detergent extracts of human leukocytes by sequential lectin affinity and gel filtration chromatography [41][42][43]. Trace levels of contaminating eosinophil peroxidase (EPO) were then removed by passage over a sulfopropyl Sephadex column [43] addition, the reaction mixture was left for 10 minutes for reaction completion and absorbance spectra were then recorded from 300 to 700 nm.…”

A predictive kinetic model for inhibitory effect of nitrite on myeloperoxidase catalytic activity towards oxidation of chloride

“…Assessment of MPO-cat n of chloride and other halides is a complex and multifunctional process [24,29]. MPO catalytic activity is dependent on initial concentrations of MPO, H 2 O 2 , Cl -, pH, H 2 O 2 to MPO concentration ratio and order of mixing.…”

“…Additionally, the model was able to estimate values for k and have distinct impact on the heme iron microenvironment [29]. Chloride occupied one site as substrate and nitrite occupied the other site as inhibitor.…”