All aerobic biological systems, including N2-fixing root nodules, are subject to 02 toxicity that results from the formation of reactive intermediates such as H202 and free radicals of 02. H202 may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN Oxygen plays a critical but incompletely understood role in the metabolism of N2-fixing root nodules. Nodule function requires a delicate balance between respiratory 02 demand and the hazards of 02 toxicity. These hazards arise from the 02 sensitivity of nitrogenase and from the production of reactive intermediates such as H202 and superoxide free radicals (02*). A key defense against 02 toxicity is superoxide dismutase, an enzyme which has been detected in many aerobic organisms, including nodule host cells (21) and bacteroids (6). The superoxide dismutase reaction produces H202 which can also be damaging and must be removed through the action of catalase or peroxidase. Although catalase is present in soybean nodules (11,21), most peroxide removal probably occurs through a coupled series of oxidation-reduction reactions involving ASC3 and glutathione (5, Fig. 1 content of nodules and the activity of the first two enzymes in this system are positively correlated with nitrogenase activity and leghemoglobin content during the early stages of nodule development (5). ASC peroxidase and DHA reductase activities are not present in isolated bacteroids, thus implying these enzymes are of plant origin (5).All three of the enzymes in this system in chloroplasts have been purified and characterized (1,12,14), but very little information is available on the corresponding enzymes in root nodules. A peroxidase from soybean root nodules was characterized by Puppo et al. (20), but the effectiveness of ASC as an electron donor was not investigated. Legume root nodules reportedly contain numerous peroxidase isozymes (16), but this may be misleading because leghemoglobins are capable of very high pseudo-peroxidase activity in the presence of appropriate reductants (20).We present evidence in this report of the presence of ASC peroxidase and associated enzymes in nine species of legumes and in red alder (Alnus rubra). ASC peroxidase from soybean root nodules was purified to near homogeneity and characterized with regards to substrate specificity, substrate affinity, absorption spectra, and effects of inhibitors. The enzyme is compared with other peroxidases, especially the ASC peroxidase from spinach chloroplasts.

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup + phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase Eco RI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn 5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn 5 -mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.