IDENTIFICATION OF A GLOBIN FREE-RADICAL IN EQUINE MYOGLOBIN TREATED WITH PEROXIDES

Reaction of metmyoglobin (MetMb) with H2O2 generates a ferryl species which is analogous to Compound II of peroxidases and is one oxidising equivalent above the initial level. The second oxidising equivalent from the peroxide is rapidly transferred into the surrounding protein generating a protein radical. Previous studies have suggested that this radical is centered on an aromatic residue, probably a tyrosine, but have not allowed unambiguous assignment. The identity of this radical has been investigated using stopped flow EPR. Reaction of equine MetMb with equimolar H2O2 gives a 6-line signal with g 2.0044 and coupling constants approx. alpha-H 2.26 and alpha-2H 0.75 mT, together with a second, broad, underlying signal. Both signals decay rapidly. The parameters of the multiplet signal are consistent with the presence of a sterically constrained tyrosine phenoxyl radical. This radical reacts further with O2, H2O2 and a number of other substrates. This activity, together with the observation that such a species is not detected with MetMb which has been iodinated solely at the Tyr-103 residue, suggests that this species is being generated from the readily accessible Tyr-103 residue, which is near the haem centre, rather than the more distant, buried, Tyr-146 residue. Reaction of this phenoxyl radical with oxygen gives the tyrosine peroxyl radical which has been previously trapped with the spin trap DMPO.