Abstract
Reaction of H2O2 with ferric leghemoglobin (metLb, the monomeric, oxygen-carrying, heme protein from root nodules of nitrogen-fixing plants) has been previously shown to generate an iron(IV)-oxo (ferryl) species and at least one protein radical. The latter has been suggested to be a tyrosine-derived phenoxyl radical present at Tyr-133 in the soybean protein and Tyr-138 in the lupin protein. To obtain further information 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 out with soybean metLb. Evidence has been obtained for at least two additional 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 (.CR1R2R3); these radicals may be side chain- or alpha-carbon-derived, their exact sites have not been determined. Some of these radicals are on the protein surface and may be key intermediates in the formation of protein dimers. These radicals have been shown to be capable of reacting with peribacteroid membrane fractions, with the consequent generation 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 protein surface to neighboring membranes may be of considerable biological significance; the destruction of this membrane is one of the earliest observable events in root nodule senescence and is associated with the loss of nitrogen-fixing activity.
Highlights
Reaction of H2O2 with ferric leghemoglobin has been previously shown to generate an iron(IV)-oxo species and at least one protein radical
Incubation of 500 M metLb with 1 mM H2O2 in the presence of the spin trap N-t-butyl-␣-phenylnitrone (PBN, 70 mM) at pH 7.4 under normoxic conditions, resulted in the detection, within a few minutes of initiation of the reaction, of a partially anisotropic EPR signal consisting of a number of broad lines
The results obtained in this study suggest that radical species are generated at multiple sites on the surface of the soybean metLb protein moiety on treatment with equimolar, or small excesses, of H2O2, and that these radicals can initiate peroxidative damage to peribacteroid membranes
Summary
Leghemoglobin; metLb, ferric (iron(III)) leghemoglobin; oxyLb, oxyferrous (iron(II)-O2) leghemoglobin; DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid; MNP, 2-methyl-2-nitrosopropane; PBM, peribacteroid membrane fractions; PBN, N-t-butyl-␣-phenylnitrone; POBN, ␣-(4-pyridyl-1-oxide)-N-t-butylnitrone. Previous studies have suggested that this lipid peroxidation process might result from (a) reaction of iron ions released from damaged Lb with excess peroxide and generation of free hydroxyl radicals which subsequently oxidize lipid molecules [11], (b) direct reaction of the iron(IV)-oxo (ferryl) species with membrane lipids, or (c) direct reaction of the globin radical(s) with membrane lipids and transfer of damage from the protein to the fatty acids (cf analogous suggestions with regard to oxidation initiated by hemoglobin and myoglobin/peroxide systems) [12,13,14] The first of these options is believed not to occur as loss of the heme absorption bands (and presumably degradation of the heme ring) in Lb systems exposed to low excesses of H2O2 does not occur either rapidly or to any major extent ( the formation of a green-colored species believed to be formed by alteration of the heme ring has been detected [15]). In order to examine whether there is more than one radical site present in Lb molecules exposed to H2O2 and whether and how such species might initiate peroxidation of the physiologically relevant peribacteroid membrane fraction of symbiosomes, both direct and spin trapping EPR studies, with a range of spin traps, have been carried out
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