This study investigated where nitrogen fixation occurs in soybean root nodules by tracking incorporation of the stable isotope 15N into different cellular fractions. Excised soybean nodules were exposed to an atmosphere enriched with 15N2, then fractionated into bacteroids, membrane envelopes, and soluble material. The membrane fraction—consisting of intracellular double-layered envelopes surrounding bacteroids—accumulated 15N label earliest and fastest, reaching maximum levels before the soluble fraction. Bacteroids themselves remained unlabeled even after 2 hours of exposure. The membrane fraction contained 37% lipid and 8.5% total nitrogen but showed negligible respiration and porphyrin-like absorption spectra. Aging nodules and those treated with CO, N2O, or H2 showed differential inhibition patterns, with CO selectively blocking transfer of fixed nitrogen to the soluble fraction while N2O completely inhibited fixation in all fractions. These findings suggest the primary site of nitrogen fixation is located in the membrane envelope structures rather than in the bacteroid cells themselves, though the bacteroid surface cannot be entirely excluded given contaminating cell-wall material in the membrane fraction.

Key findings

• 15N label first accumulated in the membrane envelope fraction within 15 minutes, before appearing in significant amounts in the soluble fraction, while bacteroid fractions remained unlabeled
• The membrane fraction contained 37% lipid and showed absorption spectra consistent with porphyrin compounds, suggesting it is primarily composed of intracellular membrane envelopes rather than plant mitochondria
• In aging (6-week-old) nodules, nitrogen fixation in the membrane fraction occurred without transfer to the soluble fraction, indicating this transfer step is the first to fail during senescence
• CO at 0.002 atm inhibited transfer of fixed nitrogen products to the soluble fraction while decreasing membrane labeling only to one-third of control levels, suggesting differential inhibition of fixation steps
• Results indicate the primary site of nitrogen fixation is in the intracellular membrane envelopes, not in the bacteroid cells themselves, challenging the previous assumption about the bacterial role in fixation