Spin trapping and protein cross-linking of the lactoperoxidase protein radical

Lactoperoxidase (LPO) reacts with H2O2 to sequentially give two Compound I intermediates: the first with a ferryl (Fe-IV=O) species and a porphyrin ra dical cation, and the second with the same ferryl species and a presumed pr otein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-n itrosobenzenesulfonic acid to give a 1:1 protein-bound radical adduct, Furt hermore, LPO undergoes the H2O2-dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in eac h of two LPO molecules, The dimer retains full catalytic activity and react s to the same extent with the spin trap, indicating that the spin trap reac ts with a radical center other than Tyr-289. The monomeric protein recovere d from incubations of LPO with H2O2 is fully active but no longer forms dim ers when incubated with H2O2, clear evidence that it has also been structur ally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H2O2-dependent oligomerization, Analysis of the interface in the LPO dimers indicates that the same protein surface is invo lved in LPO dimerization as in the normal formation of myeloperoxidase dime rs, Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates.