Further studies on a particulate enzyme preparation from nodules of soybean plants.

In previous publications (4, 6), the preparation and properties of a particulate enzyme complex from Rihizobiumn japonicutm cells isolated from the nodules of soybean plants (Glycine max Merr.) have been described. It has been established that the particles catalyze the reduction of nitrate to nitrite and that either DPNH or succinate serves as effective electron donor. Evidence indicates that the sequence of electron transport from DPNH involves an unidentified factor (replaced by menadione), a cytochrome, the Slater factor, nitrate reductase, and nitrate. When succinate is utilized, electron transfer appears to take place via succinic dehydrogenase, a cytochrome, and then a pathway from cytochrome to nitrate common with that utilized in reduced diphosphopyridine nucleotide (DPNH) oxidation. Investigations (5) on the possible physiological role of the nodule-nitrate reductase complex have provided considerable eviidence that the enzyme activity, as measured by the rate of nitrate reduction, is correlated positively with the capacity, or with factors associated with the capacity, to fix atmospheric nitrogen. In general the hemoglobin content of nodules from plants subjected to various treatments and the nitrate reductase activity of Rhizobium japoniicum cells obtained from nodules of the same plants are positively correlated. Since there is no evidence that nitrate is involved in the nitrogen fixing process, it has been postulated (4, 5) that the enzyme complex may be non-specific for oxidant and that some unidentified substance with the appropriate oxidation-reduction potential may serve as the natural oxidant under physiological conditions. In view of the physiological experiments reported (5) and the experimental evidence of Bergersen and Wilson (3) concerning the role of nodule hemoglobin in the nitrogen fixing process, the possibility has been considered that some form of nodule hemoglobin may serve as an electron acceptor for the complex. In this paper we report results of experiments in which oxygen, nodule hemiglobin, and nodule oxy-