Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation.

The soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.

Acetylene Reduction by Symbiosomes and Free Bacteroids from Broad Bean (Vicia faba L.) Nodules (Role of Oxalate).

We report the presence of oxalate in the organic acid fraction of broad bean (Vicia faba L.) nodule cytosol. Using both high-performance liquid chromatography and enzymic assays, high levels of oxalate were detected (70.4 [plus or minus] 2.4 mM). To study the potential role of oxalate as an energy-yielding substrate for nitrogenase activity, free bacteroids were isolated from nodules and found to oxidize oxalate in support of C2H2 reduction under O2 tensions that were lower than those required to oxidize succinate, another dicarboxylate commonly detected in legume nodules. Symbiosomes of broad bean, isolated for the first time from amide-producing nodules, were provided with [14C]oxalate and found to have uptake kinetics with a lower affinity [Km(oxalate) = 330 [mu]M] than that for free bacteroids [Km(oxalate) = 130 [mu]M]. In anaerobic preparations of symbiosomes supplied with purified oxyleghemoglobin, O2 consumption was stimulated by oxalate from 20.2 [plus or minus] 0.8 nmol O2 min-1mg-1 protein to 24.5 [plus or minus] 1.1 nmol O2 min-1 mg-1 protein but always remained lower than the rate of O2 consumption in free bacteroids (32.2 [plus or minus] 1.4 nmol O2 min-1 mg-1 protein). Under these conditions, C2H2 reduction activity was 9.7 [plus or minus] 0.8 and 15.1 [plus or minus] 0.9 nmol C2H4 min-1 mg-1 protein for symbiosomes and bacteroids, respectively. These data support the suggestion that oxalate may play a role as a carbon substrate in support of N2 fixation in broad bean nodules.

Absence of synproportionation between oxy and ferryl leghemoglobin. off.

The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur, at least under conditions where this process could be clearly demonstrated with myoglobin and hemoglobin. In contrast, a cross synproportionation can occur between oxyleghemoglobin and ferryl myoglobin or between ferryl leghemoglobin and oxymyoglobin. The non-exposure, at the surface of the leghemoglobin molecule, of the nearest tyrosine residue to the heme group could explain this behaviour. Thus leghemoglobin per se does not appear to be able to act as an antioxidant in removing H2O2 by synproportionation. However, in the presence of ascorbate and/or glutathione which can reduce ferryl leghemoglobin, this hemoprotein could act as an H2O2-removing antioxidant, in a process similar to that described for myoglobin. This could also explain why, despite the absence of synproportionation, ferryl leghemoglobin is not detected in nodule extracts.

Nitrogen Fixation in French-beans in the Presence of Nitrate: Effect on Bacteroid Respiration and Comparison with Nitrite.

Bacteroids isolated from French-bean (Phaseolus vulgaris L.) nodules exhibited a limited C(2)H(2) reduction activity, associated with a decline in their respiration rate, when the plants were previously fed for 24 h with a 3.5 mM Ca(NO(3))(2) solution. Similar effects were observed when bacteroid incubations received increasing nitrite concentrations. The level of cyt-c reduction recorded by a rapid spectrometric technique was increased by 15 to 20 % for bacteroids isolated from plants receiving nitrate or when N0(2)(-) was added to incubations. At the bacteroid level, the close analogy between the effects observed after supplying legumes with nitrate and the direct addition of nitrite suggested the involvement of nitrite in the inhibition of nitrogen fixation.

Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction.

Malate oxidation supported C(2)H(2) reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O(2) tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (K(m) = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (K(m) = 2.32 millimolar). Both enzymes exclusively required NAD(+) as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N(2) fixation was demonstrated. The inhibition of O(2) consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O(2), is important in a general strategy of this legume to maintain N(2) fixation under O(2) limited conditions.

Nitrite and nitric oxide as inhibitors of nitrogenase from soybean bacteroids.

Nitrite was able to strongly inhibit C(2)H(2) reduction by nitrogenase from soybean bacteroids, whereas H(2) evolution was unaffected under the same conditions. NO inhibited both C(2)H(2) reduction and H(2) evolution; during C(2)H(2) reduction, sensitivity of nitrogenase to NO was higher than to NO(2), and the K(i) values were, respectively, 0.056 and 0.52 mM. Production of NO resulting from a reduction of NO(2) by dithionite in nitrogenase incubations was observed. However, the characteristics of inhibitions and the low level of NO generated by nitrite reduction ruled out the suggestion concerning a direct role of NO to explain the inhibitory effect of NO(2) on nitrogenase.

Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions.

Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r(2) = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Psi(nod)) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C(2)H(2) reduction capacity of mildly water stressed nodules (Psi(nod) = -1.2 megapascals) was possible by increasing the external O(2) partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O(2) diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N(2) fixation activity with glucose than with succinate as an energy-yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O(2), and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N(2) fixation under water restricted conditions.