Probing nitrite coordination in horseradish peroxidase by resonance Raman spectroscopy: Detection of two binding sites.
Nitrite is a powerful oxidant that affects the activity of peroxidases towards various substrates and leads to heme macrocycle modifications in members of the peroxidase family, such as the horseradish peroxidase (HRP). We have applied resonance Raman spectroscopy to investigate the structural properties of the species formed in the reaction of NO2-with the ferric form of HRP. Our data demonstrate that the heme nitrovinyl group is partially formed at near neutral pH, without coordination of NO2-to the heme Fe. Nitrite coordinates to the heme Fe at acidic pH in the nitro binding mode, characterized by the detection of the ν(Fe-NO2) at 563cm-1, δ(FeNO2) at 822cm-1and νsym(NO2) at 1272cm-1. The sensitivity of the vibrations of the heme Fe-nitro complex to H/D exchange indicates H-bonding interaction of the heme-bound ligand with the distal environment that determines the NO2-binding mode. A model describing the different modes of NO2-binding in HRP is presented.
Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus.
Androulla
A
Ioannou
Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus. Electronic address: effiep@ucy.ac.cy.
Eftychia
E
Pinakoulaki
D001665:Binding Sites; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods*
D006420:Hemeproteins; D009573:Nitrites; D006735:Horseradish Peroxidase
Heme proteins; Nitrite; Peroxidases; Raman spectroscopy