Reaction of horseradish peroxidase with azide and some implications for the heme environmental structure. NMR and kinetic studies.
The azide complex of horseradish peroxidase was studied by high resolution 1H and 15N NMR spectroscopy and by the temperature-jump method. The heme peripheral methyl proton peaks and the ligand 15N resonance were resolved to show that binding of azide by horseradish peroxidase occurs only in acidic solution below pH 6.5. It was also found that the chemical exchange rate of azide with the ferric enzyme was much faster on the 1H and 15N NMR time scale. This was further substantiated by kinetics of azide binding by horseradish peroxidase where the chemical exchange rate was confirmed to be in the microseconds range at pH 5.0 and 23 degrees C. This rate is salient in usual ligand exchange reactions in hemoproteins so far reported. pH dependences of the first order association and dissociation rate constants were also studied by the temperature-jump method to suggest a strong linkage of the azide binding with a proton uptake of an amino acid residue on the enzyme. These results were compared with the case of horse metmyoglobin and were interpreted to indicate that a heme-linked ionizable group on the enzyme facilitates the fast entry of the ligand to the coordination site. A histidyl residue is a possible candidate for the ionizable group of the enzyme.
D001386:Azides / Q000494:pharmacology; D006418:Heme; D006735:Horseradish Peroxidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010544:Peroxidases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013696:Temperature
D001386:Azides; D006418:Heme; D006735:Horseradish Peroxidase; D010544:Peroxidases