This study examined oxygen consumption and ATP production in Rhizobium japonicum bacteroids from soybean root nodules. The researchers identified two distinct terminal oxidase systems with different oxygen affinities. A high-affinity oxidase system, sensitive to N-phenylimidazole and carbon monoxide inhibition, operates optimally at very low dissolved oxygen concentrations (0.01-0.1 μM). A low-affinity system becomes dominant at higher oxygen concentrations (≥1 μM) and is insensitive to these inhibitors. Critically, the high-affinity pathway produced five times more ATP than the low-affinity pathway at low oxygen concentrations. When purified oxyleghaemoglobin was added to bacteroid suspensions, it relieved diffusion limitations and maintained respiration at much lower oxygen concentrations where nitrogenase activity was maximal. This leghaemoglobin-facilitated oxygen delivery created an environment where bacteroid oxygen concentration remains in the optimal range for both ATP production and nitrogen fixation. The results suggest that leghaemoglobin's primary role in root nodules is facilitating oxygen diffusion while maintaining concentrations that maximize efficient ATP production through the high-affinity oxidase pathway.

Key findings

• Two terminal oxidase systems operate in soybean bacteroids: a high-affinity system (active at 0.01-0.1 μM O₂, inhibited by N-phenylimidazole and CO) and a low-affinity system (active at ≥1 μM O₂, insensitive to inhibitors)
• The high-affinity oxidase pathway produces up to five times more ATP than the low-affinity pathway at low oxygen concentrations
• Leghaemoglobin relieves diffusion limitations on bacteroid respiration and sustains oxygen delivery at concentrations optimal for nitrogenase activity and ATP production
• Maximum ATP concentration in bacteroids occurs at 0.02-0.1 μM free oxygen during leghaemoglobin deoxygenation
• Leghaemoglobin facilitates oxygen diffusion to bacteroids, enabling respiration at much lower oxygen concentrations than would otherwise be possible