Evidence for the high-spin heme iron in both stable and unstable reduced forms of lactoperoxidase: low-temperature magnetic circular dichroism data.

The unstable and stable ferrous lactoperoxidase at pH 6.0, 7.0 and 10.2 have been analysed using optical absorption and variable temperature MCD spectroscopy. The evidence is given that two high-spin forms of ferrous. LPO are always observed when the enzyme is reduced in a buffer-glycerol mixture at low temperature (ca. -20 degrees C) at which no spectral changes are seen for a long time after the reduction. Form 1 (the absorption band, 450 nm) dominates significantly over form 2 (the absorption band, 435 nm), but a relative content of form 2 increases on lowering the pH value. An annealing of the unstable LPO at high temperatures is followed by complete irreversible conversion of form 1 to form 2. In addition, at least one low-spin ferrous form exists in temperature-dependent equilibrium with the high-spin form(s) in both stable and unstable ferrous LPO. The reversible increase of its content is observed at least down to 140 K, suggesting that minor structural changes are sufficient for reaching the heme iron by a distal amino acid residue (presumably a histidine).