Reaction of ferric leghemoglobin with H 2O 2: formation of heme-protein cross-links and dimeric species

Abstract

Ferric leghemoglobin in the presence of H 2O 2 is known to give rise to protein radicals, at least one of which is centred on a tyrosine residue. These radicals are quenched by at least two processes. The first one involves an intramolecular heme-protein cross-link probably involving the tyrosine radical; this leads to the formation of a green compound with spectral characteristics differing markedly from those of ferryl and ferric leghemoglobin. This green compound cannot be reduced by dithionite or ascorbate, precluding any role for this species as an oxygen carrier. It exhibits modified EPR and pyridine haemochromogen spectra, indicating that alterations occur at the porphyrin macrocycle level. The additional compound previously described [Puppo, A., Monny, C. and Davies, M.J. (1993) Biochem. J. 289, 435–438] appears to be a mixture of ferryl Lb and this green compound. The second quenching route results in the formation of intermolecular cross-links and hence dimeric forms of the protein. Ascorbatte and glutathione inhibit both this intermolecular dimer formation and the formation of the intramolecular haem-protein cross-links and are likely to play a protective role in vivo.