Effects of concentrations of substrates supplied to N₂-fixing soybean bacteroids in flow chamber reactions

A liquid reaction medium containing dissolved air, oxyleghaemoglobin and low concentrations (0-2 mM) of glucose, succinate, malate or ethanol, was supplied to bacteroids prepared from soybean root nodules and confined in a stirred reaction chamber. Measurements of concentrations of O$_{2}$, CO$_{2}$ and NH$_{3}$ from N$_{2}$ fixation, at various flow rates through the chamber, were used to calculate rates of respiration and N$_{2}$ fixation. Glucose was utilized poorly under all conditions. Ethanol was required at 0.2-2.0 mM before stimulations of O$_{2}$ demand and N$_{2}$ fixation were observed. Demand for O$_{2}$ was enhanced by 0.05 mM to 0.5 mM succinate and malate at concentrations of O$_{2}$ from 10 nM to 50 nM. However, the concentrations of C4-dicarboxylate and dissolved O$_{2}$ determined whether N$_{2}$ fixation was stimulated or inhibited by these substrates. It is postulated that these effects were associated with the presence in the bacteroids of two malic enzymes and a pyruvate dehydrogenase complex, the resulting acetyl-CoA being channelled through the tricarboxylic acid (TCA) cycle (N$_{2}$ fixation stimulated) or to poly-$\beta $-hydroxybutyrate (PHB) accumulation (N$_{2}$ fixation inhibited). Contrary to earlier results, the O$_{2}$ consumption data suggested that there was a single terminal oxidase (K$_{\text{m}}$ 20-26 nM and V$_{\max}$ 20-29 nmol O$_{2}$ min$^{-1}$ (mg dry mass)$^{-1}$ operating in the range of O$_{2}$ concentration in which N$_{2}$ fixation occurred.