Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase.

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.