DIRECT DETECTION OF A GLOBIN-DERIVED RADICAL IN LEGHEMOGLOBIN TREATED WITH PEROXIDES

The root nodules of leguminous plants contain an oxygen-carrying protein which is somewhat similar to myoglobin. Reaction of the Fe3+ form of this protein (metleghaemoglobin; MetLb) with H2O2 is known to generate a ferryl [iron(IV)-oxo] species. This intermediate, which is analogous to Compound II of peroxidases and ferryl myoglobin, is one oxidizing equivalent above the initial level. In the present study it is shown that the second oxidizing equivalent from the peroxide is rapidly transferred into the surrounding protein, generating a protein radical which has been detected by e.p.r. spectroscopy; this reaction is analogous to that observed with metmyoglobin. An identical protein-derived species is observed with all three forms of MetLb tested (a, c1, c3) and with a number of other hydroperoxides and two-electron oxidants. This latter result, the observation that the concentration of this species is not affected by certain hydroxyl-radical scavengers, and the loss of the radical when the oxy or deoxy forms are used, demonstrate that this species is formed by electron transfer within the protein rather than by the generation and subsequent reaction of hydroxyl radicals (and related species from the other hydroperoxides). The e.p.r. signal of this species, which decays rapidly with a half-life of approx. 40 s, is consistent with the formation of a sterically constrained tyrosine-derived phenoxyl radical; protein-iodination experiments lend support to this assignment. Reaction between the radical and a number of other compounds has been observed, demonstrating that it is at least partially exposed on the surface of the protein. Analysis of the protein structure suggest that the radical may be centred on a tyrosine residue present at position 132 in the protein; this residue is close to the haem prosthetic group, which would facilitate rapid electron transfer.