Identification of the site of the globin-derived radical in leghaemoglobins.

Reaction of the Fe3+ form of the oxygen-carrying protein leghaemoglobin (MetLb), derived from the root nodules of lupins, with H2O2 is shown to generate, in addition to an iron (IV)-oxo (ferryl) species, a globin radical. This radical has been detected by EPR spectroscopy and is analogous to the species previously observed with the soybean protein. Analysis of the hyperfine coupling constants and g value of the EPR signal, together with computer simulations and the similarity of the observed spectra of that detected with the soybean form suggest that this species is also a tyrosine-derived phenoxyl radical; this species is believed to arise via an electron-transfer process within the protein with an electron being transferred from the tyrosine residue to an initially-generated Compound-1-type species. Comparison of the protein sequences and structures of the two proteins show that there is only one conserved tyrosine residue (at position 133 in the soybean and 138 in the lupin); this is believed to be the site of the phenoxyl radical. The lupin phenoxyl radical reacts with added water-soluble antioxidants and reducing agents which result in repair of the radical; this may be an important protective mechanism in vivo. Analysis of molecular models of the protein structures is in accord with both the assignment of the radical to this conserved tyrosine residue and the observed radical reactivity.