LEGHEMOGLOBIN-DERIVED RADICALS - EVIDENCE FOR MULTIPLE PROTEIN-DERIVED RADICALS AND THE INITIATION OF PERIBACTEROID MEMBRANE DAMAGE

Reaction of H2O2 with ferric leghemoglobin (metLb, the monomeric, oxyg en-carrying, heme protein from root nodules of nitrogen-fixing plants) has been previously shown to generate an iron(IV) oxo (ferryl) specie s and at least one protein radical, The latter has been suggested to b e a tyrosine-derived phenoxyl radical present at Tyr-133 in the soybea n protein and Tyr-138 in the lupin protein. To obtain further informat ion on these protein radicals and their potential interaction with the physiologically important peribacteroid membrane (which surrounds the microsymbiont in vivo), EPR spin trapping studies have been carried o ut with soybean metLb. Evidence has been obtained for at least two add itional protein derived radicals in addition to the phenoxyl radical; these radicals are transient and reactive in nature, These species are carbon-centered, and at least one is a tertiary species ((.)CR(1)R(2) R(3)); these radicals may be side chain- or alpha-carbon-derived, thei r exact sites have not been determined, Some of these radicals are on the protein surface and may be key intermediates in the formation of p rotein dimers. These radicals have been shown to be capable of reactin g with peribacteroid membrane fractions, with the consequent generatio n of lipid-derived radicals. The formation of such radicals may result in the depletion of membrane antioxidants and the initiation of lipid peroxidation, This transfer of damage from the heme center via the pr otein surface to neighboring membranes may be of considerable biologic al significance; the destruction of this membrane is one of the earlie st observable events in root nodule senescence and is associated with the loss of nitrogen-fixing activity.