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The cytosol from root nodules of soybean, bean, and cowpea contained Fe and Cu capable of catalyzing the formation of highly reactive free radicals. Specific and sensitive assays based on free radical-mediated DNA degradation revealed that most catalytic Fe and Cu were present as small chelates (300-600 Da). The involvement of catalytic Fe in free radical production during nodule senescence, which was induced by exposure of plants to continuous darkness for 2-4 days, was investigated. (i) Free heme remained at a constant and low concentration (1-4% of total nodule heme) during senescence, indicating that it is not an important constituent of the catalytic Fe pool of nodules. (ii) Catalytic Fe of nodule cytosol promoted deoxyribose degradation and linolenic acid peroxidation in reaction mixtures containing physiological concentrations of ascorbate and H2O2. Deoxyribose degradation but not lipid peroxidation required hydroxyl radicals to proceed. (iii) The cytosol from senescent nodules, particularly of bean and cowpea, sustained in vitro higher rates of deoxyribose degradation and lipid peroxidation than the cytosol from unstressed nodules. Both degradative processes were inhibited by the Fe chelator desferrioxamine and were correlated with the content of catalytic Fe in the nodule cytosol. (iv) Although other transition metals (Cu, Mn, Mo, and Ni) were present in significant amounts in the low molecular mass fraction (<3 kDa) of the nodule cytosol, Fe is most likely the only metal involved in free radical generation in vivo. (v) By using dimethyl sulfoxide as a molecular probe, formation of significant amounts of hydroxyl radical was observed in vivo during senescence of bean and cowpea nodules.